MASS SPECTROMETRY METHODS FOR THE ANALYSIS OF BIODEGRADABLE HYBRID MATERIALS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Ahlam Alalwiat August, 2015
MASS SPECTROMETRY METHODS FOR THE ANALYSIS OF BIODEGRADABLE HYBRID MATERIALS Ahlam Alalwiat Dissertation Approved: Advisor Dr. Chrys Wesdemiotis Committee Member Dr. Leah Shriver Committee Member Dr. Adam W. Smith Committee Member Dr. Sailaja Paruchuri Accepted: Department Chair Dr. Kim C. Calvo Dean of the College Dr. Chand K. Midha Interim Dean of the Graduate School Dr. Rex D. Ramsier Date Committee Member Dr. Matthew L. Becker ii
ABSTRACT This dissertation focuses on the characterization of hybrid materials and surfactant blends by using mass spectrometry (MS), tandem mass spectrometry (MS/MS), liquid chromatography (LC), and ion mobility (IM) spectrometry combined with measurement and simulation of molecular collision cross sections. Chapter II describes the principles and the history of mass spectrometry (MS) and liquid chromatography (LC). Chapter III introduces the materials and instrumentation used to complete this dissertation. In chapter IV, two hybrid materials containing poly(t-butyl acrylate) (PtBA) or poly(acrylic acid) (PAA) blocks attached to a hydrophobic peptide rich in valine and glycine (VG2), as well as the poly(acrylic acid) (PAA) and VG2 peptide precursor materials, are characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS) and ion mobility mass spectrometry (IM-MS). Collision cross-sections and molecular modeling have been used to determine the final architecture of both hybrid materials. Chapter V investigates a different hybrid material, [BMP-2(HA)2], comprised of a dendron with two polyethylene glycol (PEG) branches terminated by a hydroxyapatite binding peptide (HA), and a focal point substituted with a bone morphogenic protein mimicking peptide (BMP-2). MALDI-MS, ESI-MS and IM-MS have been used to iii
characterize the HA and BMP-2 peptides. Collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) have been employed in double stage (i.e. tandem) mass spectrometry (MS/MS) experiments to confirm the sequences of the two peptides HA and BMP-2. The MALDI-MS, ESI-MS and IM-MS methods were also applied to characterize the [BMP-2(HA)2] hybrid material. Collision cross-section measurements and molecular modeling indicated that [BMP-2(HA)2] can attain folded or extended conformation, depending on its degree of protonation (charge state). Chapter VI focuses on the analysis of alkyl polyglycoside (APG) surfactants by MALDI-MS and ESI-MS, MS/MS, and by combining MS and with ion mobility (IM) and/or ultra-performance liquid chromatography (UPLC) separation in LC-IM and LC- IM-MS experiments. Chapter VII summaries this dissertation s findings. iv
ACKNOWLEDGEMENTS First and foremost, I would like to thank my God (Allah) for everything. When I was five years old, my dream was to become mathematics scientist. After more than 25 years, I achieved my dream to be research scientist and that would not have possible without the efforts of the people I would like to thank here. I would like to express my special appreciation and thanks to my advisor Dr. Chrys Wesdemiotis for his help, assistance, guidance and support in developing and completing my research work and studies at The University of Akron. I would like to thank you for allowing me to grow as a research scientist. Your advice has been priceless. I would like to thank my committee members, Dr. Leah Shriver, Dr. Adam W. Smith, Dr. Sailaja Paruchuri and Dr. Matthew L. Becker for serving as my committee members. I am most appreciative for their time, precious assistance and invaluable comments. I would like also to thank and acknowledge the Department of Chemistry at The University of Akron, both faculty and staff, for their assistance. I would like to thank the Ministry of Higher Education of the Kingdom of Saudi Arabia for supporting me in my Ph.D. studies. v
I would like to thank my former colleagues; Dr. Nadrah Alawani, Dr. Aleer Yol, Dr. Bryan Katzenmayer, Dr. Vincenzo Scionti, Dr. Chunxiao Shi, Dr. Xiumin Liu, Dr. Kai Guo, Dr. Marisa Garchedi as well as the current group members; Sahar Sallam, Michelle Kushnir, Lydia Cool., Sarah Robinson, Nicolas Alexander, Selim Gerislioglu, Mehmet Atakay, Jialin Mao, Kevin Endres and Dr. Ivan Dolog for their camaraderie, support, friendship and helpful discussions. I want to thank Dr. Xinqiao Jia, Dr. Sarah E. Grieshaber, Bradford A. Paik (from University of Delaware), Dr.Wen Tang, Dr. Mark D. Foster, Jacob Hill, Mengmeng Yao, Dr. George R. Newkome, Dr. Charles N Moorefield, Dr. Xiaocun Lu, Dr. Xiaopeng Li, Dr. Christina Mastromatteo, Dr. Alper Buldum, and Dr. Tarab Ahmad and for all the input, advice and help they provided me. I would like to thank my small family, especially my husband (Hatam) and my daughters (Zahra, Fatima, Zainab and Hawra) for their love and support. I am grateful to my big family; my parents, sisters, brothers and my husband s family for their support. Finally, I would like to thank all my teachers and friends for their support. vi
TABLE OF CONTENTS Page LIST OF TABLES....xii LIST OF FIGURES.....xiv LIST OF SCHEMES....xxvi ACRONYMS AND ABBREVIATIONS...xxvii CHAPTER I. INTRODUCTION....1 II. INSTRUMENTAL METHODES BACKGROUND...7 2.1 Mass Spectrometry.....7 2.2 Ionization Methods....9 2.2.1 Electrospray Ionization (ESI)....10 2.2.2 Matrix Assisted Laser Desorption/Ionization (MALDI)......13 2.3 Mass Analyzers....14 2.3.1 Quadrupole Mass Analyzer......16 2.3.2 Time-of-Flight Mass Analyzer......18 2.3.3 Quadrupole Ion Trap Mass Analyzer....22 2.3.4 Quadrupole/Time-of-Flight (Q/ToF) Mass Analyzer....25 2.4 Detectors.......27 vii
2.5 Tandem Mass Spectrometry (MS/MS).....29 2.6 Ion Mobility Mass Spectrometry (IM-MS)......31 2.7 Liquid Chromatography Mass Spectrometry (LC-MS)....38 III. MATERIALS AND INSTRUMENTATION..45 3.1 Materials........ 45 3.2 Instrumentation.........46 3.2.1 HCT Ultra II ESI-QIT Mass Spectrometer..47 3.2.2 Ultraflex III ToF/ToF Mass Spectrometer 48 3.2.3 Synapt HDMS Ion Mobility Mass Spectrometer...49 3.2.4 Acquity UPLC....50 IV. TOP-DOWN MASS SPECTROMETRY OF HYBRID MATERIALS WITH HYDROPHOBIC PEPTIDE AND HYDROPHILIC POLYMER BLOCKS...51 4.1 Background....51 4.2 Sample Preparation and Instruments Used.........54 4.2.1 Sample Preparation for MALDI-ToF/ToF-MS......54 4.2.2 Sample Preparation for ESI-QIT-MS.........56 4.2.3 Sample Preparation for ESI-Q/ToF-MS..... 57 4.2.4 VG2 Peptide with Surfactant.......58 4.3 Characterization of VG2 peptide.....59 4.3.1 Characterization of VG2 by MALDI-ToF/ToF-MS and MS 2...61 4.3.2 Characterization of VG2 by ESI-QIT-MS and ESI-ToF/ToF-MS and MS n...66 4.3.3. Characterization of VG2 by using surfactant as a solvent...75 4.4 Characterization of PAA Polymer......79 viii
4.4.1 Characterization of PAA by MALDI-ToF/ToF-MS.... 81 4.4.2 Characterization of PAA by ESI-Q/ToF-MS....... 82 4.5 Characterization of PtBA-VG2.......85 4.5.1 Characterization of PtBA-VG2 by MALDI-ToF/ToF-MS.....86 4.5.2 Characterization of PtBA-VG2 by ESI-Q/ToF-MS........88 4.6 Characterization of PAA-VG2....89 4.6.1 Characterization of PAA-VG2 by MALDI-ToF/ToF-MS.....91 4.6.2 Characterization of PAA-VG2 by ESI-Q/ToF-MS.........92 4.7 Structural Information from Collision Cross-Sections.........95 4.7.1 Drivation of Collision Cross-Sections from Traveling Wave IM- MS Experiments......95 4.7.2 Molecular Modeling.....106 4.8 Conclusions......114 V. MASS SPECTROMETRY CHARACTERIZATION OF BIOACTIVE PEPTIDE- SYNTHETIC POLYMER CONJUGATES.....115 5.1 Background... 115 5.2 Sample Preparation and Instruments Used.......118 5.2.1 Sample Preparation for MALDI-ToF/ToF-MS...118 5.2.2 Sample Preparation for ESI-QIT-MS... 119 5.2.3 Sample Preparation for ESI-Q/ToF-MS.......120 5.3 Characterization of BMP-2 Peptide...... 121 5.3.1 Characterization of BMP-2 by MALDI-ToF/ToF-MS......122 5.3.2 Characterization of BMP-2 by ESI-QIT-MS and ESI-Q/ToF- MS.122 5.4 Characterization of HA Peptide.....135 ix
5.4.1 Characterization of HA by MALDI-ToF/ToF-MS.....136 5.4.2 Characterization of HA by ESI-QIT-MS and ESI-Q/ToF- MS. 139 5.5 Characterization of BMP-2(HA)2 hybrid material... 149 5.5.1 Characterization of BMP-2(HA)2 by MALDI-ToF/ToF- MS...151 5.5.2 Characterization of BMP-2(HA)2 by ESI-Q/ToF-MS....153 5.6 Collision Cross-Sections and Molecular Modeling.. 155 5.7 Conclusions 165 VI. ANALYSIS OF ALKYL POLYGLYCOSIDE (APG) SURFACTANTS USING POLARITY VS. SHAPE SENSITIVE MULTIDIMENSIONAL MASS SPECTROMETR..... 166 6.1 Background.... 166 6.2 Experimental Procedures....... 169 6.2.1 MALDI-ToF/ToF-MS.......169 6.2.2 ESI-QIT-MS........170 6.2.3 ESI-Q/ToF-MS.....170 6.2.4 Ultra-Performance Liquid Chromatography (UPLC)... 171 6.3 Characterization of APGs by MALDI-ToF/ToF-MS.....173 6.3.1 MALDI-MS of Alkyl Polyglucosides (APGs).... 173 6.3.2 MALDI-MS 2 of Alkyl Polyglucosides (APGs).... 176 6.4 Analysis of the Alkyl Polyglucosides by ESI-QIT-MS and ESI-Q/ToF- MS....180 6.4.1 ESI-MS of alkyl polyglucosides (APGs)... 180 6.4.2 ESI-MS 2 of Alkyl Polyglucosides (APGs)..... 188 x
6.5 ESI-IM-MS and ESI-IM-MS 2 of Alkyl Polyglucosides (APGs)...192 6.6 UPLC-MS and UPLC-IM-MS studies of Alkyl Polyglucosides (APGs)...208 6.7 Conclusions.....214 VII. SUMMARY..216 REFERENCES.....219 APPENDICES...241 APPENDIX A. ADDITIONAL DATA....242 APPENDIX B. COPYRIGHT PERMISSION...279 xi
LIST OF TABLES Table Page 4.1 Drift time and collision cross-section data of singly and doubly protonated polyalanine oligomers, H-(Ala)n-OH.....97 4.2 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of Figure 4.30....98 4.3 Experimental collision cross-sections of nine doubly and triply protonated PtBAn-VG2 oligmers, derived using the calibration plot of Figure 4.30.....101 4.4 Experimental collision cross-section of twelve doubly and triply protonated PAAn-VG2 oligomers, derived using the calibration plot of Figure 4.30.104 4.5 Collision cross-sections of doubly and triply protonated of PtBA-VG2 oligomers. The experimental CCSs were obtained from Table 4.3. The calculated values are the average CCSs of the 50 energy-minimized structures, obtained using projection approximation (PA), exact hard sphere scattering (EHSS), and trajectory (TJ) methods of the MOBCAL program..110 4.6 Collision cross-sections of doubly and triply protonated of PAA-VG2 oligomers. The experimental CCSs were obtained from Table 4.4. The calculated values are the average CCSs of the 50 energy-minimized structures, obtained using projection approximation (PA), exact hard sphere scattering (EHSS), and trajectory (TJ) methods of the MOBCAL program...112 5.1 Drift time of BMP-2(HA)2 ions for IM-MS analysis at traveling wave height and traveling wave velocity of 8V and 300 m/s, respectively....155 5.2 Corrected collision cross-section of the polyalanine calibrant ions, [H(Ala)nOH+zH] +2 (z=1-2), deduced from drift times measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8V...157 xii
5.3 Comparison of collision cross-sections determined using the calibration curve in Figure 5.33 with published collision cross-section for ubiquitin and equine cytochrome C ions with different numbers of proton charges....158 5.4 Collision cross-sections of different multiply charged ions of BMP-2(HA)2...159 5.5 Collision cross-sections calculated from energy-minimized structures of BMP- 2(HA)2 using the projection approximation (PA), trajectory (TJ), and exact hard sphere scattering (EHSS) methods of the MOBCAL programare...160 6.1 Calculated mass values for the [APG+Na]+ ions of APGs mass ([GmCn+Na]+) = n * 162.05275 + m* 14.01565 + 22.9898 (Na + ) + 18.01055...176 xiii
LIST OF FIGURES Figure Page 2.1 Generic instrument schematic of a mass spectrometer...8 2.2 (A) Droplet formation at the needle tip of the ESI capillary; (B) decomposition of a droplet in the electrospray source according to Rayleigh s equation...10 2.3 Schematic of the Coulombic explosion of a charged droplet...12 2.4 Illustration of the MALDI process 14 2.5 Schematic representation of the quadrupole mass analyzer..16 2.6 Schematic of a linear ToF mass analyzer..19 2.7 Schematic of a reflectron ToF mass analyzer 20 2.8 Schematic of the delayed extraction principle for ToF mass analyzer..21 2.9 Schematic of the quadrupole ion trap mass analyzer.23 2.10 Stability diagram for a QIT, in which four singly charged ions with the masses m4< m3< m2< m1 were injected. If no DC is used (U=0 and az =0), ion trajectories are determined only by the RF field (qz). Ions with qz <0.908 remain trapped (m₁, m₂, m₃) and ions with qz >0.908 are ejected (m₄)......25 2.11 Schematic of the quadrupole/time-of-flight (Q/ToF) Mass analyzer.26 2.12 Schematic of a microchannel plate detector and the electron multiplication within the channels...27 2.13 Schematic of a Daly detector..28 2.14 Schematic of a tandem mass spectrometry in-space and in-time...30 xiv
2.15 Schematic of the Waters Synapt HDMS Q/ToF mass spectrometer 2006 Waters Corporation.32 2.16 Schematic of the triwave section of the Synapt HDMS system.33 2.17 Schematic of a stacked ring ion guide...34 2.18 Schematic of the operation of a traveling wave ion guide containing ring electrodes for transferring the ions through the buffer gas.....35 2.19 Schematic of the basic components of an HPLC-MS system.....40 2.20 Van Deemter plot (dashed line) and individual plots of the terms of the Van Deemter equation....43 3.1 Schematic view of the Bruker HCT ultra II ESI-QIT mass spectrometer..46 3.2 QIT with external nci source for ETD experiments in the HCTultra II QIT mass spectrometer 47 3.3 Schematic view of the Acquity UPLC system 2004 Waters Corporation..50 4.1 Nomenclature of peptide fragment ions according to Biemann.......59 4.2 (A) MALDI-MS spectrum of sodium cationized VG2 peptide acquired using the sandwich method. The peptide was dissolved in DMSO at 20 mg/ml. CHCA (20 mg/ml) matrix was mixed with NaTFA salt in the ratio (100:10, v/v %). B) MALDI-MS spectrum of VG2 peptide mixed with CHCA without using solvents 60 4.3 Structures of XVPGVGVPGVGX and XGVGPVGVGPVX.....61 4.4 MALDI-MS 2 spectrum of sodium cationized of VG2 peptide (m/z 1090.0) acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum. X designates the propargyl-substituted end groups of the peptide (138 Da at the N-terminus and 111 Da at the C-terminus), which allow it to react with azide-terminated polymer to form a peptide-polymer hybrid material...62 4.5 MALDI-MS spectrum of XVG2 acquired using a 20 mg/ml DMSO solution of the peptide and the sandwich method. CHCA (20 mg/ml) matrix mixed with NaTFA salt in the ratio (100:10, v/v %) was used for the bottom and top layers...63 4.6 MALDI-MS 2 spectrum of sodium cationized of XVG2 peptide (m/z 966.025) acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide xv
is shown on top of the spectrum. X designates the propargyl-substituted end groups of the peptide.....64 4.7 (A) ESI-MS spectrum of VG2 peptide dissolved, in ammonium acetate buffer (ph=6.64) at 0.01 mg/ml plus 10 % (v %) of MeOH acquired with the QIT mass spectrometer. B, C) Expanded views of the m/z regions containing B) doubly charged ions (orange box) and C) singly charged ions (purple box)......65 4.8 CAD (A) and ETD (B) mass spectra of the doubly sodiated VG2 ([VG2+2Na] +2 at m/z 556.7) acquired on the ESI-QIT mass spectrometer. The ETD ion reaction time was 400 ms. All fragments are singly sodiated unless marked by which denates sodiation plus one H/Na exchange (two Na+ions overall)...67 4.9 CAD-MS 3 spectrum of the [VG2 +Na] + fragment (m/z 1090) from [VG2+2Na]+ 2 acquired on the ESI-QIT mass spectrometer......68 4.10 CAD-MS 3 spectrum of the y9 (m/z 854.5) fragment from [VG2+2Na]+² (m/z 556.7) acquired on the ESI-QIT mass spectrometer...69 4.11 CAD-MS 3 spectrum of the a6 (m/z 640.3) fragment from [VG2+2Na] +2 (m/z 556.7) acquired on the ESI-QIT mass spectrometer...70 4.12 ETD spectrum of [VG2+H+Na] +2 (m/z 545.6) acquired on the ESI-QIT mass spectrometer, using an ion-ion reaction time of 600 ms. All fragments are singly sodiated unless massed by H, which denotes ions with proton charges (no Na + )...71 4.13 ETD spectrum of doubly potassiated VG2 ([VG2+2K] +2 at m/z 572.6) acquired on the ESI-QIT mass spectrometer, using an ion-ion reaction times of 600 ms. An asterisk indicates singly charged ions with two K+ (one K+ charge and one H/K exchange..72 4.14 Plausible structures of the ETD fragments from doubly charged VG2. Note that c6 is formed by cleavage of bonds within a proline residue..73 4.15 ESI-MS spectrum of VG2 dissolved in ammonium acetate buffer (ph=6.64) at 0.05 mg/ml + 1 % (v %) of MeOH, acquired with the Synapt Q/ToF mass spectrometer 74 4.16 A) 2-D IM-MS plot (m/z vs. drift time) of VG2 dissolved in ammonium acetate buffer (ph=6.64) at 0.01 mg/ml with 10 % (v %) MeOH, acquired with the Synapt Q/ToF mass spectrometer. B) Mass spectrum extracted from IM region @ of the 2-D diagram. C) Mass spectrum extracted from IM region $ of the 2-D diagram. D) Drift time chromatogram (mobilogram) for [VG2+Na] + (m/z 1090.58)....75 4.17 A) ESI-MS spectrum of VG2 dissolved at the concentration of 0.05 mg/ml in aqueous 0.01 mg/ml of n-dodecyl-α-d-maltoside (α-c₁₂g₂) containing 10 % (v %) MeOH. The spectrum was acquired with the Synapt Q/ToF mass xvi
spectrometer B) Expanded view of the m/z region of doubly charged ions (green box). C) Expanded view of the m/z region of singly charged ions (purple box)....76 4.18 2-D IM-MS 2 plot (m/z vs. drift time) of [VG2+Na] + (m/z 1090), acquired with a Synapt Q/ToF mass spectrometer from a VG2 solution (0.05 mg/ml) in surfactant containing 10 % (v %) MeOH.......77. 4.19 Mass spectrum extracted from the 2-D IM-MS 2 plot (m/z vs. drift time) of sodium cationized VG2 (m/z 1090), formed by ESI of a 0.05 mg/ml solution of the peptide in 0.01 mg/ml aqueous surfactant containing 10% (v %) MeOH...78 4.20 MALDI-MS spectrum of polyacrylic acid (PAA) in positive ion mode. The sample was dissolved in THF/ MeOH (50:50, v/v %) at 20 mg/ml. SA matrix (20 mg/ml) and NaTFA cationizing salt (10 mg/ml) solutions were mixed (100:10, v/v %) and the sanswich method was used.....80 4.21 MALDI-MS spectrum of polyacrylic acid (PAA) in negative ion mode. The sample preparation was the same in positive mode.....81 4.22 2-D IM-MS plot (m/z vs. drift time) of PAA dissolved in THF/MeOH (50:50, v/v %) at 0.1 mg/ml plus 1% (v %) formic acid, acquired with the Synapt Q/ToF mass spectrometer in positive ion mode. B) Mass spectrum extracted from the circled mobility region, where [PAA+2K] +2 ion dominate...83 4.23 2-D IM-MS plot (m/z vs. drift time) of diazide-terminated polyacrylic acid in negative ion mode. The sample was dissolved in THF/MeOH (50:50, v/v %) at 0.1 mg/ml plus 1% (v %) formic acid was added. B) Mass spectrum extracted from the circled region of -3 ions [PAA-3H+K] -3 anions dominate. C) Mass spectrum extracted from the circled region of -2 ions, where [PAA-2H] -2 anions dominate...84 4.24 MALDI-MS spectrum of poly(tert-butyl acrylate)-vg2 peptide] (PtBA-VG2) hybrid material acquired on the MALDI-ToF/ToF mass spectrometer in positive mode. The sample was dissolved in NH4OAc/ MeOH (50:50, v/v %) at 20 mg/ml. DCTB (20 mg/ml) and KTFA (10 mg/ml) were mixed in the ration 100:10 (v/v %) and the sandwich method was used...87 4.25 2-D IM-MS plot (m/z vs. drift time) of the PtBA-VG2 hybrid material. The sample was dissolved in NH4OAc/ MeOH (50:50, v/v %) at 0.1 mg/ml plus 1% (v %) formic acid. The mass spectra extracted from the +2 and +3 mobility regions are depicted beside and below the plot, respectively.89 4.26 MALDI-MS spectrum of polyacrylic acid-vg2 peptide (PAA-VG2) hybrid material acquired on the MALDI-ToF/ToF mass spectrometer in positive ion mode. The sample was dissolved in DMSO at 20 mg/ml; CHCA served as xvii
matrix and the sandwich method was used. An designates the number of PAA units and Bn the number of PtBA units......90 4.27 MALDI-MS spectrum of the PAA-VG2 hybrid material acquired on the MALDI-ToF/ToF mass spectrometer in negative ion mode. The sample was dissolved in DMSO at 20 mg/ml; CHCA served as matrix and the sandwich method was used. An designates the number of PAA units and Bn the number of PtBA units...92 4.28 2-D IM-MS plot (m/z vs. drift time) of PAA-VG2 hybrid material in positive ion mode. The sample was dissolved in NH4OAc at 0.01 mg/ml and plus 10 % (v %) MeOH. The mass spectra extracted from the region of doubly and triply charged PAA-VG2 are shown next to the plot....93 4.29 2-D IM-MS plot (m/z vs. drift time) of PAA-VG2 hybrid material in negative ion mode. The sample was dissolved in NH4OAc at 0.01 mg/ml plus 10 % (v %) MeOH was added. The mass spectra extracted from the regions of doubly and triply charged PAA-VG2 are shown next to the plot........94 4.30 Plot of corrected drift times (arrival times) against corrected published cross sections for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V 96 4.31 IM-MS chromatogram (drift time distribution) of the ions at m/z 1054 and m/z 1182 from PtBA-VG2. The insets show the mass spectra extracted from the main peaks.........99 4.32 IM-MS chromatogram of the ions at m/z 1030 and m/z 1102 from PAA-VG2. The insets show the mass spectra extracted from the center components....102 4.33 IM-MS chromatogram of the ions at m/z 807 and m/z 831 from PAA-VG2. The insets show the mass spectra extracted from the right components peaks...103 4.34 Plot of experimental collision cross-section vs. m/z for PtBA-VG2 and PAA-VG2 oligomers with (a) +2 and (b) +3 protoneted charges...105 4.35 Plot of calculated collision cross-section (PA method) vs. relative energy for 50 energy-minimized structures of a) [PtBA₆-VG2] +2 (m/z 1054.1), and b) [PtBA7 -VG2] +2 (m/z 1118.2) with linear architecture (orange circle), or cyclic architecture (blue triangle). On average, the cyclic architectures are more stable (by ~ 2.9 kcal/mol). Representative linear ans cyclic structures are shown in the lower right and upper left corners, respectively......107 4.36 Plot of calculated collision cross-section (PA method) vs. relative energy for 50 energy-minimized structures of a) [PAA7-VG2] +2 (m/z 921.97), and b) [PAA10-VG2] +2 (m/z 1029) with linear architecture (orange circle), or cyclic xviii
architecture ( blue triangle). On average, the cyclic architectures are more stable (by ~ 4 kcal/mol). Representative linear ans cyclic structures are shown in the lower right and upper left corners, respectively...108 4.37 Plot of calculated collision cross-section (PA method) for PtBA-VG2 oligomers with linear and cyclic structures (a) +2 or (b) +3 charges, and experimental collision cross-section of the same oligomers vs. m/z ratio......111 4.38 Plot of calculated collision cross-sections (PA method) PAA-VG2 oligomers with linear and cyclic structures (a) +2 or (b) +3 charges, and experimental collision cross-section of the same ions vs. m/z ratio.........113 5.1 MALDI-MS spectrum of BMP-2 peptide dissolved in H2O at 20 mg/ml. CHCA (20 mg/ml) served as matrix and the sandwich method was used...121 5.2 MALDI-MS 2 spectrum of protonated BMP-2 (m/z 2118.500) acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum. All b and a fragments in this Figure are of the type bn+1, yn+1 and an+1(radical ions)......123 5.3 A) ESI-MS spectrum of BMP-2 peptide dissolved in H2O at 0.01 mg/ml + 10 % (v %) of MeOH acquired with QIT mass spectrometer. B-D) Expanded views of the m/z regions of B) quadruply charged ions (green box), C) triply charged ions (red box), and D) doubly charged ions (blue box) of BMP-2 peptide.124 5.4 ESI-CAD mass spectrum of the doubly protonated BMP-2 peptide, [BMP-2+2H] +2 (m/z 1059.6), acquired on the ESI-QIT mass spectrometer...125 5.5 ESI-ETD mass spectrum of the doubly protonated BMP-2 peptide, [BMP-2+2H] +2 (m/z 1059.6), acquired on the ESI-QIT mass spectrometer.126 5.6 ESI-CAD mass spectrum of the triply protonated BMP-2 peptide, [BMP-2+3H] +3 (m/z 706.8), acquired on the ESI-QIT mass spectrometer... 127 5.7 ESI-ETD mass spectrum of the triply protonated BMP-2 peptide, [BMP-2+3H] +3 (m/z 706.6), acquired on the ESI-QIT mass spectrometer... 128 5.8 ESI-ETD mass spectrum of the quadruply charged BMP-2 peptide, [BMP-2+3H+ K] +4 (m/z 539.8), acquired on the ESI-QIT mass spectrometer. An asterisk indicates that the ion contains K +...129 5.9 ESI-MS³ mass spectrum acquired on the ESI-QIT mass spectrometer by CAD of [BMP-2+3H+K] +3 (m/z 719.6), formed by ETD of [BMP-2+3H+K] +4 (m/z 539.8). An asterisk indicates that the ion contains K +...130 xix
5.10 ESI-MS spectrum of BMP-2 peptide acquired with the Synapt Q/ToF mass spectrometer. The peptide was dissolved in H2O at 0.01 mg/ml and 10% of MeOH + 0.5% formic acid (both v/v %) were added to this solution to enhance the ESI efficiency....131 5.11 2-D IM-MS plot (m/z vs. drift time) of BMP-2 dissolved in H2O at 0.05 mg/ml plus 10% MeOH and 0.5% formic acid (both v/v %), acquired with the Synapt Q/ToF mass spectrometer..... 132 5.12 IM-MS chromatograms (drift time distributions) for (a) [BMP-2+2H] +2 (m/z 1059.5720) and (b) [BMP-2+3H] +3 (m/z 706.6887)...133 5.13 ESI-MS 2 spectrum of [BMP-2+3H] +3 (m/z 706.76), acquired with the Synapt Q/ToF mass spectrometer. Singly charged fragments are shown in red color and doubly charged fragments in green color......134 5.14 MALDI-MS spectrum of HA peptide dissolved in H2O at 20 mg/ml. CHCA (20 mg/ml) served as matrix and the sandwich method was used...135 5.15 MALDI-MS 2 spectrum of protonated HA peptide, [HA+H]+ (m/z 1412.6), acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum, together with the bond cleavages giving rise to the yn series and to b14.....137 5.16 MALDI-MS 2 spectrum of [HA+O+H]+ (m/z 1428.668) acquired on the MALDI- ToF/ToF mass spectrometer. Scheme on top of the spectrum shows the product formed after elimination of the oxidized Met side substituent and the consecutive bond cleavages leading to sequence-indicative fragments...138 5.17 ESI-MS spectrum of HA peptide dissolved in H2O at 0.01 mg/ml + 10% MeOH + 0.5% formic acid (both v/v %), acquired with QIT mass spectrometer 139 5.18 ESI-CAD mass spectrum of doubly protonated HA peptide, [HA+2H] +2 at m/z 706.9, acquired on the ESI-QIT mass spectrometer...140 5.19 ESI-CAD spectrum of the y8 ion (m/z 869.5) in the ESI mass spectrum of HA peptide (Figure 5.17), acquired on the ESI-QIT mass spectrometer. The same spectrum obtained by an ESI-MS 3 experiment on the y8 ion formed by CAD of [HA+2H] +2 (Figure 5.18). The fragments observed support the composition [GMKPSPRP+H] +. 141 5.20 ESI-MS 3 spectrum of the y5 ion (m/z 553.3) from [HA+2H] +2 (Figure 5.18), acquired on the ESI-QIT mass spectrometer. The same spectrum is obtained by CAD of the y5 ion present in the ESI mass spectrum of HA (Figure 5.17). The fragments observed corroborate the composition [PSPRP+H] +...142 xx
5.21 ESI-ETD mass spectrum of triply protonated HA peptide, [HA+3H] +3 (m/z 471.6), acquired on the ESI-QIT mass spectrometer....143 5.22 ESI-ETD mass spectrum of the doubly protonated y10 ion (m/z 528.1) in the ESI mass spectrum of HA (Figure 5.17), acquired on the ESI-QIT mass spectrometer.......143 5.23 ESI-MS spectrum of HA peptide dissolved in H2O at 0.01 mg/ml + 10% (v/v %) MeOH, acquired with the Synapt Q/ToF mass spectrometer..144 5.24 2-D IM-MS plot (m/z vs. drift time) of HA dissolved in H2O at 0.01 mg/ml plus 10% (v/v %) MeOH, acquired with the Synapt Q/ToF mass spectrometer..145 5.25 (a) 2-D IM-MS plot (m/z vs. drift time) of m/z 1412.8 from HA peptide after selection by the Q mass analyzer. (b) 1-D drift time distribution (chromatogram) extracted from this plot......146 5.26 Mass spectra extracted from each of the three peaks in the drift time chromatogram of m/z 1412.8 from HA peptide....147 5.27 ESI-MS 2 spectrum of [HA+3H] +3 (m/z 471.6341), acquired with the Synapt Q/ToF mass spectrometer.....148 5.28 (a) 2-D IM-MS2 plot (m/z vs. drift time) of [HA+2H] +2 (m/z 706.9) from HA peptide, acquired with the Synapt Q/ToF mass spectrometer. (b) 1-D drift time chromatogram of m/z 706.9. MS 2 was performed by CAD after the IM separation step...149 5.29 Mass spectrum extracted from the 2-D IM-MS 2 plot of [HA+2H] +2 (m/z 706.9) from HA peptide.......150 5.30 MALDI-MS spectrum of BMP-2(HA)2 hybrid material dissolved in H2O at 10 mg/ml. SA (20 mg/ml) was served as matrix and the sandwich method was used. The structure of BMP-2 (HA)2 is shown on top of the spectrum....152 5.31 ESI-MS spectrum of BMP-2(HA)2 hybrid material dissolved in H2O at 0.01 mg/ml + 30% of MeOH + 0.5% formic acid (both v/v %), acquired with the Synapt Q/ToF mass spectrometer...153 5.32 (a) 2-D IM-MS plot (m/z vs. drift time) of BMP-2(HA)2 hybrid material dissolved in H2O at 0.01 mg/ml + 30% MeOH + 0.5% formic acid (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. (b) Drift time chromatograms of intact BMP-2(HA)2 charge states....154 5.33 Plot of corrected drift times (arrival times) against corrected published cross sections for the +1 and +2 charge of polyalnine oligomers. Drift times were xxi
measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8 V....156 5.34 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with +4 proton charges at the following sits: Triazoles (N, N); HA1 (R); HA2 (R). The average relative energy and CCS (PA) of all 50 candidate structures are 29 kcal/mol and 790 Ų, respectively. A representative structure is inserted inside the plot 162 5.35 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with +5 proton charges at the following sits: BMP-2 (K, K); HA1 (K, R); HA2 (R). The optimized structures can be divided into two conformational families (circles and squares) with average relative energy and CCSs (PA) of 44 kcal/mol and 887 Ų (circles) and 116 kcal/mol and 1058 Ų (squares), respectively. Representative structures for each population are inserted inside the plot.163 5.36 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with +6 proton charges at the following sits: BMP-2 (K, K); Triazole (N); HA1 (K, R); HA2 (R). The optimized structures can be divided into two conformational families (circles and squares) with average relative energy and CCSs (PA) of 61 kcal/mol and 868 Ų (circles) and 144 kcal/mol and 1163 Ų (squares), respectively. Representative structures for each population are inserted inside the plot.164 6.1 MALDI-MS spectrum of 818 UP alkyl polyglucoside using sandwich method and DIT as matrix. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆... 172 6.2 MALDI-MS spectrum of 1200 UP alkyl polyglucoside using sandwich method and DIT as matrix. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆......173 6.3 MALDI-MS spectrum of 2000 UP alkyl polyglucoside using sandwich method and DIT as matrix. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆......174 6.4 Nomenclature of glyco-conjugate fragment ions......177 6.5 MALDI-MS 2 spectrum of [G₆C₁₂Na]+ ion at m/z 1181.564 from 818 UP sample...178 xxii
6.6 MALDI-MS 2 of 857.445 [G₄ C₁₂Na+] ion at m/z 857.445 from 1200 UP sample......178 6.7 ESI-MS spectrum of 818 UP alkyl polyglucoside dissolved in H2O at 0.01 mg/ml + 10% MeOH + 2% LiTFA (both v/v %), acquired with QIT mass spectrometer. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI. The number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆....181 6.8 ESI-MS spectrum of 1200 UP alkyl polyglucoside dissolved in H2O at 0.01 mg/ml + 10 % MeOH + 2 % LiTFA (both v/v %), acquired with QIT-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI. The number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆......182 6.9 ESI-MS spectrum of 2000 UP alkyl polyglucoside dissolved in H2O at 0.01 mg/ml + 10 % MeOH + 2 % LiTFA (both v/v %), acquired with QIT-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI. The number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆......183 6.10 ESI-MS spectrum of 818 UP alkyl polyglucoside dissolved in H2O at 0.05 mg/ml + 10 % MeOH + 1 % NaTFA (both v/v %), acquired with Q/ToF-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI... 185 6.11 ESI-MS spectrum of 1200 UP alkyl polyglucoside dissolved in H2O at 0.05 mg/ml + 10 % MeOH + 1 % NaTFA (both v/v %), acquired with Q/ToF-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI.....186 6.12 ESI-MS spectrum of 2000 UP alkyl polyglucoside dissolved in H2O at 0.05 mg/ml + 10 % MeOH + 1 % NaTFA (both v/v %) acquired with Q/ToF-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI....187 6.13 ESI-MS 2 spectrum of G₁C₁₂Li+ (m/z 354.2) from the 1200 sample acquired on the ESI-QIT mass spectrometer... 188 6.14 ESI-MS 2 spectrum of G₂C₈Na+ (m/z 477.2) from the 2000 sample acquired on the ESI-QIT mass spectrometer...189 6.15 ESI-MS 2 spectrum of G₃C₈Na+ (m/z 639.3) from the 818 sample acquired on the ESI-QIT mass spectrometer...190 xxiii
6.16 ESI-MS 2 spectrum of the G₁ DI Li+ (m/z 703.5) from the 1200 sample acquired on the ESI-QIT mass spectrometer...191 6.17 2-D IM-MS plot (m/z vs drift time) of 818 UP dissolved in H2O + 10% MeOH at 0.05 mg/ml + 1 % NaTFA (both v/v %) acquired with the Synapt Q/ToF mass spectrometer. 192 6.18 Mass spectrum extracted from the [APG] +1 mobility region in the ESI-IM-MS plot of 818 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)12, # = (CH₂)₁₄, $ = (CH₂)₁₆, and % = (CH₂)₁₈..193 6.19 Mass spectrum extracted from the [X-mer] +1 mobility region in the ESI-IM-MS plot of the 818 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn........194 6.20 2-D IM-MS plot (m/z vs. drift time) of the 1200 UP dissolved in H2O + 10% MeOH at 0.05 mg/ml + 1% NaTFA (both v/v %), acquired with the Synapt Q/ToF mass spectrometer.....195 6.21 Mass spectrum extracted from the [APG] +1 mobility region in the ESI-IM-MS plot of 1200 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)12, # = (CH₂)₁₄, $ = (CH₂)₁₆, and % = (CH₂)₁₈...195 6.22 Mass spectrum extracted from the [X-mer] +1 mobility region in the ESI-IM-MS plot of 1200 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn...196 6.23 2-D IM-MS plot (m/z vs. drift time) of 2000UP APGs samples dissolve in H2O +10 % MeOH at 0.05 mg/ml +1 % NaTFA (both v/v %) acquired with the Synapt Q/ToF mass spectrometer.........197 6.24 Mass spectrum extracted from the [APG] +1 mobility region in the ESI-IM-MS plot of 2000 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)12, # = (CH₂)₁₄, $ = (CH₂)₁₆, and % = (CH₂)₁₈....197 6.25 Mass spectrum extracted from the [X-mer] +1 mobility region in the ESI-IM-MS plot of 2000 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn...198 6.26 Total ion intensities of the dimer ions detected from the 818 UP sample, plotted against the corresponding APG concentrations....199 xxiv
6.27 2-D IM-MS plot (m/z vs. drift time) of n-dodecyl-α-d-maltoside (α-c₁₂g₂) dissolved in H2O + 10% MeOH at 0.05 mg/ml + 1% NaTFA (both v/v %), acquired with the Synapt Q/ToF mass spectrometer........200 6.28 Mass spectrum extracted for +1 band in the ESI-IM-MS plot of n-dodecyl-α-dmaltoside (α-c₁₂g₂).....201 6.29 Mass spectrum extracted for +3 band in the ESI-IM-MS plot of n-dodecyl-α-dmaltoside (α- G₂C₁₂).....201 6.30 Mass spectrum extracted from the +5 band in the ESI-IM-MS plot of n-dodecyl -α-d- maltoside (α-c₁₂g₂)....202 6.31 ESI-MS 2 spectrum of the APG dimer ion at m/z 663 (from 818 UP) at energy of 4 ev (trap collision cell), followed by IM separation of the MS 2 products. The 2-D IM-MS2 plot is shown on the top and the extracted spectrum on the bottom. The sample was dissolved in water at 0.05 mg/ml plus 1% (v %) MeOH and the data were acquired with the Synapt Q/ToF mass spectrometer....204 6.32 2-D IM-MS 2 plot (top) and extracted spectrum (bottom) of the APG dimer ion at m/z 719 from 1200 UP. The collision energy was 10 ev..205 6.33 2-D IM-MS 2 plot (top) and extracted spectrum (bottom) of the APG dimer ion at m/z 635 from 2000 UP. The collision energy was10 ev....207 6.34 UPLC MS chromatogram (TIC vs. time) of APG samples. Gradient elution as percentage of mobile phase B [methanol/isopropanol (50:50), (v/v %)]: linear increase from 60% to 70% over 1 min, from 70% to 80% over 1 min, and from 80% to 90% over 1 min. The mobile phase flow rate was 250 μl min 1. The retention times are marked....208 6.35 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #1 from the 818 UP APG sample....210 6.36 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #2 from the 818 UP APG sample... 211 6.37 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #3 from the 818 UP APG sample... 212 6.38 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #4 from the 818 UP APG sample... 213 xxv
LIST OF SCHEMES Scheme Page 4.1 Synthesis of azide-functionalized PAA.....79 4.2 Synthesis of azide-functionalized PtBA-VG2 and PAA-VG2...86 5.1 Synthetic procedure to BMP-2(HA)2...151 6.1 General fragmentation pathways of APGs upon MALDI. The Bb fragment is an internal fragment, but indistinguishable from B-type fragments because the non-reducing end is not substituted.....179 xxvi
ACRONYMS AND ABBREVIATIONS MS m/z MS n SEC GC CE LC ESI MALDI MS 2 CAD CID HPLC RP-LC IMS PAA PtBA VG2 Mass Spectrometry mass to charge ratio Multiple Stages of mass analysis Size Exchange Chromatography Gas Chromatography Capillary Electrophoresis Liquid Chromatography Electrospray Ionization Matrix Assisted Laser Desorption/Ionization Tandem Mass Spectrometry Collisionally Activated Dissociation Collision Induced Dissociation High Performance Liquid Chromatography Reverse Phase Liquid Chromatography Ion Mobility Spectrometry Poly Acrylic Acid Poly t-butyl Acrylate VPGVGVPGVG peptide xxvii
PtBA-VG2 PAA-VG2 PEG HA HA peptide BMP-2 Poly t-butyl acrylate block attached to VPGVGVPGVG peptide Poly acrylic acid block attached to VPGVGVPGVG peptide Polyethylene Glycol Hydroxyapatite Bioactive Hydroxyapatite binding peptide Bone Morphogenic Protein-2 BMP-2 peptide Bioactive peptide that mimics the Bone Morphogenic protein-2 BMP-2(HA)2 UPLC IM EI CI FAB FD ppm IT Q DC rf Q/ToF QIT ToF ToF/ToF Hybrid materials attached to two HA peptide and BMP-2 peptide Ultra-performance liquid chromatography Ion Mobility Electron Ionization Chemical Ionization Fast Atom Bombardment Field Desorpation Parts Per Million Ion Trap Quadrupole Direct Current radiofrequency Quadrupole Time of Flight Quadrupole Ion Trap Time of Flight Tandem Time of Flight xxviii
DE MCP ETD TWIG K CCS EDC NP-LC UHPLC TWIMS CHCA SA DCTB DIT Β-C₈G₁ Delayed Extraction Microchannel Plate Electron Transfer Dissociation Traveling Wave Ion Guide Ion Mobility Constant Collision Cross-Section Enhanced Duty Cycle Normal Phase Liquid Chromatography Ultra High Performance Liquid Chromatography Traveling Wave Ion Mobility Spectrometry α- cyano-4-hydroxycinnamic acid 3,5-dimethoxy-4-hydroxycinnamic acid trans 2 (3 (4 tert butylphenyl) 2 methyl 2 propenyliedene)malononitrile Dithranol N-octyl-β-D-glucoside 818UP Plantacare 818 1200UP Plantaren 1200 2000UP Plantaren 2000 PMMA PS DSC PPC CD Poly Methyl Acrylate Poly Styrene Differential Scanning Calorimetry Pressure Perturbation Calorimetry Circular Dichroism Spectroscopy xxix
UV FTIR GPC NMR Ultraviolet Fourier Transform Infrared Spectroscopy Gel Permeation Chromatography Nuclear Magnetic Resonance ¹H NMR Proton Nuclear Magnetic Resonance FTICR XVG2 α-c₁₂g₂ ATRP WV WH TJ PA EHSS XRD PLA PGA PA PCL PLGA TGFb APGs PAD Fourier Transform Ion Cyclotron Resonance Mass Spectrometry VPGVGVPGVG peptide with NH2 in c-terminal N-dodecyl-α-D-maltoside Atom Transfer Radical Polymerization Wave Velocity Wave Height Trajectory method Projection Approximation method Exact Hard Sphere Scattering method X Ray Diffraction Poly Lactic Acid Poly Glycolic Acid Polyamide Poly ɛ-caprolactone Poly Lactic-co-glycolic Acid Transforming Growth Factor b Alkyl Polyglycosides Pulsed Amperometric Detection xxx
MEKC TLC ToF-SIMS Miceller Electrokinetic Chromatography Thin Layer Chromatography Time of Flight Secondary Ion Mass Spectrometry xxxi
CHAPTER I INTRODUCTION Mass spectrometry (MS) is a powerful analytical technique that converts the analyte samples to gas-phase ions, then separates them by their mass to charge ratio (m/z) and detects them. It is utilized in many fields such as synthetic polymers, proteins, peptides, oligosaccharides and new materials research due to its high sensitivity, low sample consumption, and speed of analysis. 1 MS can be used to identify the chemical structures of molecules, determine architectures, sequence biopolymers and quantitate analytes. 1,2 Nevertheless, it has some limitations that need to be overcome especially for the analysis of complex mixtures, such as copolymers, surfactants, natural products, proteins, new synthetic materials and high molecular weight polymers, which may be impossible to analyze by using traditional MS approaches. MS is also unable to distinguish between isomeric species, complex isobaric species, and species that have different charge and same m/z. To resolve these limitations, MS analysis can be extended to tandem MS, which includes multiple stages of mass analysis (MS n ), or connected to separation techniques. Chromatographic methods like size exclusion chromatography (SEC), gas chromatography (GC), capillary electrophoresis (CE), liquid chromatography (LC), or ion mobility spectrometry (IMS) can increase the sensitivity and selectivity of MS. 1,2 1
MS analysis starts with the formation of gas-phase ions from the analyte sample. Various mass spectrometric techniques are used today to classify and characterize synthetic polymers. Electrospray ionization (ESI) 3,4 and matrix assisted laser desorption/ionization (MALDI) 5-7 are soft ionization techniques and widely used in structural and compositional studies of synthetic polymers because they can form intact gas-phase ions from such molecules. 8,9 In ESI, the analyte sample is dissolved in a volatile solvent and the solution is introduced by a syringe into the mass spectrometer. 8 In MALDI, a matrix, which is a small organic compound, is used to absorb the laser light and prevent the aggregation and/or destruction of the analyte molecules. A solution of matrix is mixed with a solution of analyte then the mixture is deposited on a sample plate and is dried. A laser beam irradiates the mixture and induces ionization of the analyte. 8 Interfaces between separation and mass spectrometry techniques have been applied to characterize polymeric materials but to a much lesser extent than they have been used to analyze biomolecules. 9-11 Mass analysis of the ions formed by MALDI or ESI is called single stage mass spectrometry. Application of single stage mass spectrometry provides information about the molecular masses of the components in the polymer. From the mass of the polymer components, the number of monomer units, and the total end groups in the polymer chains can be calculated. On the other hand, single stage mass spectrometry does not provide information about polymer sequences and architectures (how a polymer s constituents are connected to each other and the overall polymer shape, respectively), and it does not distinguish between isomeric and frequently also isobaric components. Such information may be accessed by doing a second stage of mass spectrometry (tandem 2
mass) or multi-stage mass spectrometry. 12 In MS/MS, the ion of interest, called the precursor ion, is selected and activated to fragments, usually by collisions with neutral gas molecules. The precursor ions may fragment in defined spaces or at defined times to ions called product ions. This technique is known as collisionally activated dissociation (CAD) or collision-induced dissociation (CID) and it is commonly utilized to cause fragmentation of a precursor ion. The CD-generated fragments are separated according to their m/z in a second mass analyzer or by a second mass analysis step in the same mass analyzer. The experiments of tandem mass spectrometry in space are performed by using two or more mass analyzers (beam instruments), and the experiments of tandem mass spectrometry in time are performed in the same mass analyzer but at different times byusing trap instruments. 1, 12, 13 Tandem mass spectrometry has been a very helpful tool in determinations of individual end groups 13-18 and architectures of polymers. 19-20 Liquid chromatography (LC) is the most widely used technique for separating non-volatile mixtures such as proteins, surfactants, and synthetic polymers into their individual components. 21 LC has high separation power but it cannot identify unknown components when used alone with simple detection systems, such as UV detectors. Coupling LC separation techniques with MS identification techniques together creates a very powerful analytical tool with high separation power and selectivity for molecular identification. 21-23 High performance liquid chromatography (HPLC) separates the components of a mixture based on their polarity and their physical or chemical interaction with the mobile phase and stationary phase. LC has been coupled with ESI, which allows the direct introduction of eluted fractions into the ESI source where the mixture components are separated further based on their m/z ratio. 24-27 Reverse phase 3
liquid chromatography (RP-LC) is the easiest LC method to couple with ESI-MS because polar solvents are used as the mobile phase in LC, and ESI uses protic or polar solvents. 4 Ion mobility spectrometry (IMS) is an analytical technique that separates gas phase ions based on their mobility in weak electric fields. 28, 29 Ions are separated in an IMS tube according to their size and charge. 30 IMS can be coupled with mass spectrometry to separate and identify isomers, 31, 32 metabolites, 33-36 chiral compounds, 37 complex mixtures, 38-40 and polymeric conformers. 42-43 The first commercially available instrument that coupled mass spectrometry with the separation capability of IMS to permit ion mobility mass spectrometry (IM-MS) studies was the Waters Synapt HDMS mass spectrometer. 44 In this instrument, ion mobility based separation is effected in a travelling wave (T-wave) cell, located between the quadrupole (Q) and time-of-flight (ToF) analyzers of a Q/ToF tandem mass spectrometer. Ions generated in the ion source 34, 44 are separated by their mobilities in the T-wave chamber. The common theme of this dissertation is applications of multidimensional mass spectrometry, coupled with ion mobility or liquid chromatography separation techniques, for the analysis of polymers, peptides, hybrid materials and alkyl glycoside surfactants. Chapter II will introduce a brief history and the principles of mass spectrometry, ion mobility spectrometry, and liquid chromatography, providing succinct descriptions of the instrumentation and fundamentals of these techniques. Chapter III will describe the materials and instruments used to complete this dissertation. The following three chapters are research project chapters and each will be briefly introduced below. Lastly, Chapter VII summarizes the conclusions drawn from this dissertation. Following the last chapter 4
are appendices with supplementary data and copyright permissions for artwork reproduced in this dissertation. Chapter IV discusses the use of top-down mass spectrometry for the analysis of hybrid materials containing poly (acrylic acid) (PAA) or poly (t-butyl acrylate) (PtBA) blocks attached to the hydrophobic peptide (elastin mimic) VPGVGVPGVG (VG2). In this study, four samples are characterized by using MALDI-MS, ESI-MS and IM-MS. The four samples include the hydrophobic peptide (VPGVG)₂, poly acrylic acid (PAA) and two hybrid materials (PtBA-VG2 and PAA-VG2). Using IM-MS, the collision crosssections (CCSs) for the two hybrid materials are experimentally measured and compared with theoretical CCSs, which are calculated by using molecular modeling, in order to distinguish the architecture of the hybrid materials. Chapter V describes the characterization of a new hybrid material developed for use as a tissue engineering substrate in bone healing applications. This polymer-peptide based hybrid has a dendron structure with two polyethylene glycol (PEG) branches terminated by a bioactive hydroxyapatite binding peptide (HA), and a focal point substituted with a different bioactive peptide (BMP-2) that mimics the bone morphogenic protein-2. In this study, the HA peptide, BMP-2 peptide, and BMP-2(HA)2 hybrid material are characterized by MALDI-MS, ESI-MS and IM-MS, as well as tandem MS (MS 2 ) and multistage MS (MS n ). Chapter VI deals with the characterization of alkyl polyglycoside surfactant blends by MALDI-MS, ESI-MS, MS 2, and ultra-performance liquid chromatography (UPLC). MS and MS/MS are also combined with either ion mobility (IM) or IM and UPLC separation to examine the dispersion abilities of various combinations of 5
separation techniques. As will be shown in this dissertation, multidimensional dispersion greatly simplifies the resulting spectra, allowing for adequate separation and conclusive characterization of the complex materials examined. 6
CHAPTER II INSTRUMENTAL METHODS BACKGROUND 2.1 Mass Spectrometry Mass spectrometry (MS) is an analytical methods that is used widely in a variety of fields, including materials science, drug discovery, natural product chemistry, proteomics, metabolomics, pharmaceutical, environmental and forensic science. This method can be utilized to determine the structure of individual substances or complex mixtures. To obtain a spectrum, the analyte molecules are converted into gas-phase ions. The ions are then separated according to their mass-to-charge ratio (m/z). Every mass spectrometer has five main components: inlet system, ion source, mass analyzer, ion detector and data system (Figure 2.1). Mass analyzers and detectors work under high vacuum (low pressure), but the ion source can work at ambient pressure or within a high vacuum system (10 5-10 10 torr). The high vacuum is required to avoid collisions or interaction of the ions with other gaseous molecules. The sample inlet system is utilized to introduce the sample to the mass spectrometer by direct injection, direct insertion, or chromatography. For direct injection, the sample is introduced as a gas or a solution through a capillary. For direct insertion, 7
the sample is deposited on a probe, a plate, or a target. Then, it is inserted into the source, which is usually under vacuum. In the ion source, analyte molecules are converted into gas-phase ions. Then, the mass analyzer separates the ions according to their mass-to-charge ratio (m/z). The detector measures the currents of the separated ions and converts them into electronic signals. Finally, the data system records these signals 1, 45 and produces a mass spectrum. Different ionization sources and mass analyzers are utilized within the field of mass spectrometry. The ionization methods and mass analyzers used to complete this dissertation will be discussed in-depth in the following sections. Inlet system Ion source Mass analyzer Detector Vacuum system Data system Figure 2.1 Generic instrument schematic of a mass spectrometer. 8
2.2 Ionization Methods The most important and challenging step in mass spectrometric analysis the generated ions, which precedes the separation of the ions in the mass analyzer based on their m/z. 46 Mass spectrometry measurements utilize ions because it is easy to experimentally control the motion and direction of ions. 45 There are several methods to create gas-phase ions and selecting the appropriate ionization method depends on the analyte of interest. Ionization methods include charge transfer from charged species in the gas phase, electron capture, electron ejection, proton capture and proton ejection, as well as cationization of neutral species in the ion source. 1, 8 Ionization methods can be classified to two types, hard and soft. Hard ionization methods include electron ionization (EI), chemical ionization (CI), fast atom bombardment (FAB), and field desorption (FD). These were initially used in mass spectrometry to study volatile and thermally stable compounds. The intact analyte peak is not observed because hard ion sources cause the analyte to fragment. The structure of the analyte can be determined from fragment ions but the spectrum is complicated and hard to interpret. In soft ionization, the intact analyte peak is detected with minimal or no fragmentation. 1 Such methods include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). These two methods are widely used for the characterization of large biomolecules and synthetic polymers. In this chapter, only ESI and MALDI will be discussed, which were the ionization methods used to complete the studies described in this dissertation. 9
2.2.1 Electrospray Ionization (ESI) Electrospray ionization is a soft ionization method that is used to study peptides, proteins, carbohydrates, small oligonucleotides, synthetic polymers and lipids. 46 The idea of forming ions in gas phase from a solution was explained first by Malcolm Dole in 1968. 47 The modern ESI method used currently was introduced by John Fenn in the late 1980s when he demonstrated the formation of multiply charged ions from large protein molecules. 3, 4 In ESI, gas-phase ions are formed when the analyte solution is sprayed through a capillary tube at ambient pressure under the influence of a high electric field. 1 (A) ESI capillary Taylor cone Nebulizing gas flow Electrostatic field High voltage 2-6kV (B) Figure 2.2 (A) Droplet formation at the needle tip of the ESI capillary (reproduced with permission from reference 48); (B) decomposition of a droplet in the electrospray source according to Rayleigh s equation (reproduced with permission from reference 1). 10
The four main steps in the ESI process are ion formation, nebulization, desolvation and ion desorption. 1, 45, 48-50 The ions can form in solution before the nebulization step and that helps to give a high abundance of ions and good electrospray sensitivity. Some analytes do not ionize in solution, but at the surface of the spray droplets where the strong electrical charge created during nebulization, desolvation, and ion evaporation can help to ionize the analyte. In the nebulization step, a solution of the analyte is introduced in the ion source with a grounded metallic capillary tube by means of either a syringe pump or the exit of an LC column. At the same time, a nebulizing gas, usually nitrogen, surrounds the needle in the spray chamber concentrically. The droplets are produced in the end of the capillary by combining strong shear forces and the strong electrostatic field (2-6 kv) in the spray chamber. This step produces highly charged droplets from the solution. In this high electric field, the repulsion forces of the accumulated charges at the tip of the capillary become higher than the surface tension. This causes the droplet to take the shape of a Taylor cone and break into smaller droplets. 1, 45 Figure 2.2 A shows droplet formation at the needle tip of the ESI capillary and Figure 2.2B shows decomposition of a droplet in the electrospray source according to Rayleigh s equation. When the force of the Coulomb repulsion between charges becomes close to the surface tension (i.e. at the Rayleigh limit) of the droplet, the charged droplets distort. This can occur before the Rayleigh limit because the droplets are mechanically distorted by the electrical field, thus decreasing the repulsion needed to break them down. This decomposition is explained by the Rayleigh equation (Equation 2.1) where q is the charge, ε 0 is permittivity of the environment, γ is the surface tension and D is the diameter of the spherical droplet. 11
q 2 = 8π 2 ε 0 γd 3 (Equation 2.1) On their way to the mass analyzer, charged droplets pass through a heated capillary surrounded with heated dry gas (usually nitrogen). When the solvent evaporates the droplets undergo shrinkage. Figure 2.3 Schematic of the Coulombic explosion of a charged droplet (adapted with permission from reference 48). The charge-residue model and the ion-desorption model are the two main mechanisms that explain ion formation from the droplets. In the charge-residue model, the droplets become smaller because the solvent evaporates while the charge remains the same. This results in increased charge per volume unit and electrostatic tension at the surface of the droplets. When the surface tension force that holds the droplets at the Rayleigh limit becomes close to the charge repulsion forces, the droplets deform into Taylor cones and ultimately rupture into smaller droplets. This process is called Coulombic explosion (Figure 2.3). Solvent evaporation and Coulombic explosion repeat until only a single molecule with one or more charges remains. In the ion-desorption model, the charge density and the electric field on the droplet surface increase due to 12
solvent evaporation. When the electric field becomes very high but less than the Rayleigh 1, 45, 48, 49, instability limit, it can cause desorption of single ions with one or more charges. 51 2.2.2 Matrix-Assisted Laser Desorption/Ionization (MALDI) MALDI is a powerful soft ionization method which was first introduced in 1988 by Karas and Hillenkamp in Germany. Nicotinic acid was utilized as the matrix to ionize proteins. Tanaka invented a very similar method in the same year, soft laser desorption, 1, 52, 53 by using metal powder and glycerol to ionize poly (ethylene glycol). In MALDI, analyte ions remain intact in the gas phase, which simplifies the analysis of a wide range of large molecules such as proteins and polymers. The analyte is mixed with a large molar excess of a small organic compound called the matrix. Most matrices are aromatic molecules with OH or COOH groups. The matrix has many purposes. It absorbs laser energy at the laser wavelength and that helps the analyte to desorb from the surface and prevents it from forming clusters. The matrix also plays an assistant role in transferring a proton or other cationization agent (e.g. Na +, Ag +, and etc.) to the analyte and protects the analyte molecules from photo-induced decomposition. 1 Figure 2.4 illustrates of the MALDI process. There are a number of sample preparation techniques for mixing the matrix and the analyte together. They include dried-droplet, sandwich matrix and solvent-free sample preparation, and all techniques can give homogeneous crystal formation. 45 The matrix and analyte are deposited on a target. Then, the target is inserted into the vacuum after the solvents evaporate. 13
Figure 2.4 Illustration of the MALDI process (adapted with permission from reference 1). In the MALDI process, the sample-matrix crystals are bombarded with a laser, usually a UV laser (e.g., Nd:YAG laser). The matrix absorbs the energy, which leads to ionized and evaporated matrix. During this process, the matrix creates a gas-phase plume on the target surface and that helps to evaporate the analyte molecules. Finally, the analyte molecules are ionized by gas-phase proton transfer from the matrix ions or cation transfer from a cationizing agent (usually salt) added to the analyte/matrix mixture. 1 2.3 Mass Analyzers The mass analyzer is the most important component of the mass spectrometer. It is used to separate the gas-phase ions according to their mass-to-charge ratio (m/z) and it is usually held under high vacuum in order to avoid any collision between the ions and other gaseous molecules before they reach the detector. Various types of mass analyzers have been developed which can be classified in two main categories. The first category is 14
the scanning analyzer (e.g. the quadrupole mass analyzer), which separates the ions one at a time. Only the selected mass range of ions or specific ions are allowed to pass and reach the detector successively. The second category of mass analyzers can separate the ions in space (e.g. the time-of-flight tube). They disperse all the ions inside the mass analyzer and allow them to travel simultaneously to the detector. 1 Each type of mass analyzer has advantages and disadvantage based on its characteristic features such as its mass range, transmission, resolution, mass accuracy and scan speed. The mass range of the mass analyzer gives the lowest and highest m/z values that can be measured. The transmission of a mass analyzer is defined as the ratio of the number of ions reaching the detector and the number of ions entering the mass analyzer. Resolution (either m2 -m1 in Da or (m2 -m1)/m1 in ppm) is the ability of the mass analyzer to clearly separate two neighboring ions having small difference in m/z value. The mass accuracy is the mass difference between measured and theoretical m/z ratio. The last characteristic is scan speed. It is the total number of spectra that can be measured per unit 1, 23 of time. The next section will discuss the mass analysers used to complete this dissertation. These analyzers include the quadrupole, quadrupole ion trap (QIT), time-offlight (ToF), and quadrupole/time-of-flight (Q/ToF) mass analyzers. 15
Figure 2.5 Schematic representation of the quadrupole mass analyzer (adapted with permission from reference 1). 2.3.1 Quadrupole Mass Analyzer A quadrupole mass analyzer consists of four cylindrical (or hyperbolic) rods arranged parallel to each other (Figure 2.5). Ions are separated by applying an oscillating electric field between the rods. Direct current (DC), also referred as U, and radiofrequency (RF potentials), also referred as V cos ωt, are applied to all rods and each pair of opposite rods have different polarity. When positive DC and RF potentials are applied to one set of rods, the other set of rods has negative DC and RF potentials, as shown in equations 2.2-2.3: Φ = (U V cos ωt) (Equation 2.2) Φ = (U V cos ωt) (Equation 2.3) ω = 2πƒ (Equation 2.4) Where Φ is the overall potential applied to the rods, U is the DC potential, V is amplitude, t is the time and ω is the angular frequency of the RF potential. Equation 2.4 16
explains the relationship between the angular frequency and the radio-frequency f. Ions are separated by changing the DC and RF potentials and keeping their ratio the same. Under a specific setting of the U and V potentials, only specific ions (specific m/z) will oscillate through the rods and reach the detector. After the ions are formed in the ion source, a series of focusing or accelerating lenses provide the appropriate electric potentials to accelerate the ions in the direction of the z-axis and enter the mass analyzer. The potentials applied to the rods help the ions to move with a zigzag motion in the z-direction inside the quadrupole. When positive ions enter the space between the four rods, they are attracted to the negatively charged rods. To avoid ions reaching and touching the rods, the potential of these rods changes sign to become positively charged, and that change the direction of the positive ions. The scenario is the opposite for negative ions. By repeating this process, ions move in zigzag motion in the z-direction. The RF potential applied to the rods focuses the ions in the center of the quadrupole to avoid losing them by collision with the rods. The combination of DC and RF potentials creates a stability window that allows only specific ions with a specific m/z to travel inside the quadrupole, whereas other ions hit the rods and become neutralized. 1, 45, 51 The Mathieu equation (Equation 2.5) and the solution of the Mathieu equation (Equation 2.6-2.7) define the motion of the ions inside the quadrupole which is described by their x/y coordinates and the two dimensionless parameters a and q. d 2 u d 2 ( ωt 2 )2 + (a u-2q u cosωt) = 0 (Equation 2.5) a u =a x = a y = 8zeU mω 2 r 2 (Equation 2.6) 17
q u =q x = q y = 4zeV mω 2 r 2 (Equation 2.7) In these equations, u represents the x and y coordinates of the ions moving in the center of the quadrupole. The au and qu are dimensionless parameters (Equation 2.6 and 2.7) that specify the magnitude of the DC potential (U) and RF amplitude (V), respectively. The term r is one half of the distance between opposite rods. When the values of x and y stay smaller than r0, ions will have a stable trajectory through the quadrupole and will be 1, 45 successfully transmitted to the detector. 2.3.2 Time-of-Flight Mass Analyzer The time-of-flight (ToF) mass analyzer consists of a field-free flight tube. Accelerated ions are separated according to their flight time and velocity when they travel a distance L for the time t in the flight tube until they reach the detector (Figure 2.6). Pulsed laser ion sources (e.g. MALDI) are usually coupled with ToF analyzers. Ions are introduced to the ToF analyzer as packets, which are accelerated to the field-free region by applying a potential (V) between the electrode and extraction grid of the ion source. When ions with mass m and total charge (q = ze) are accelerated by using a potential equal to V s, they gain a potential energy Eel in the acceleration region which is converted to kinetic energy Ek (as seen in Equation 2.8). In other words, all ions are accelerated by the same potential (V s ) and reach the same kinetic energy (Ek), but they will have different velocities (υ) because they have different m/z (Equations 2.8-2.11), so they reach the detector at different times. 18
Figure 2.6 Schematic of a linear ToF mass analyzer (adapted with permission from reference 1). E k = ze V s = 1 2 mυ2 = E el (Equation 2.8) t = L v (Equation 2.9) υ = 2zeV s m (Equation 2.10) t = L m z 2eV s (Equation 2.11) In these equations, υ is the velocity of the ions leaving the source, m is the mass of the ion and ze is the charge of the ion. This equation also shows that the heavier ions will travel more slowly and spend longer time in the flight tube than the lighter ions. Unfortunately, ions with the same m/z value may be formed at varied locations and thus will have different initial kinetic energies. They will have different flight times, which leads to poor resolution in linear ToF instruments. This problem was solved by the 19
development of the reflectron (Figure 2.7) and delayed extraction techniques (Figure 1, 45, 51, 52 2.8). Figure 2.7 Schematic of a reflectron ToF mass analyzer (adapted with permission from reference 1). The reflectron, also called electrostatic reflector, is an electrostatic mirror that is utilized to reflect the ions and send them back through the flight tube to increase their path length inside the tube (Figure 2.7). It consists of a series of grid or ring electrodes connected through a resistive network that is placed at the end of the flight tube. Ions with higher kinetic energy will spend less time in the flight tube, because of their higher velocity, but they will travel deeper into the reflectron than ions with the same m/z but lower kinetic energy with proper adjustment of the reflctron potential. All ions with a specific m/z will reach the detector at the same time. 1, 45, 51, 52 20
Figure 2.8 Schematic of the delayed extraction principle for ToF mass analyzer (adapted with permission from reference 1). Delayed extraction (DE), also called delayed pulsed extraction and pulsed ion extraction, is another technique used to reduce the kinetic energy spread between ions with the same m/z value. The acceleration potential is not applied to the extraction region during the ionization pulse, which allows ions separation in a field-free region according to their initial kinetic energies before the extraction (i.e., acceleration) grid is pulsed. During this time, ions with higher kinetic energy are moving closer to the grid while other ions with the same m/z but less kinetic energy are moving more slowly, and thus do not come as close to the grid of the source region. During this time-delay or time-lag, the ions drift for nanoseconds to microseconds in the ion source before being accelerated (Figure 2.8). The potential energy of the slower ions will increase more than the energy of the faster ions after applying the extraction grid pulse; therefore, the slow ions will 21
have more kinetic energy to fly faster and catch up with the fast ions. Both ions will reach the detector at the same time. 1, 45, 52 2.3.3 Quadrupole Ion Trap Mass Analyzer The quadrupole ion trap (QIT) mass analyzer, also called the three-dimensional analog of a quadrupole mass filter, was developed by Paul and colleagues in 1958. 45 It is an ion-storing device consisting of three electrodes, where one ring electrode is located between two end cap electrodes (Figure 2.9). It uses oscillating electric fields to trap ions inside the electrodes and store them in space by controlling their motions. An RF potential, Φ = U-V cos ωt, is applied to the ring electrode while the two end cap electrodes are grounded. Due to the potential difference and the shape of the three electrodes, a three-dimensional hyperbolic field is generated which forces the ions to move in stable trajectories towards the trap center where they are stored and trapped. The entrance end cap has a small hole at the center that allows the injected ions to enter into the ion trap while the exit end cap has multiple holes that allows for efficient ejection of the ions to reach the detector. The ions inside the trap will carry the same charge polarity and, hence, repel each other. Their trajectories would destabilize, leading to their ejection from the trap. To avoid such ion loss, a buffer gas such as helium is introduced in the trap. The buffer gas collides with the ions to remove excess energy and pushes them back toward the trap center. 1, 45, 48 22
Figure 2.9 Schematic of the quadrupole ion trap mass analyzer (adapted with permission from reference 48). The ion motion inside the quadrupole ion trap can be described by the Mathieu equation and the dimensionless parameters q z and q r (Equation 2.12). No DC field is used in modern QITs (a z and U are equal to 0 in Equation 2.13). As mentioned above, the ions in the quadrupole mass analyzer travel in the z direction after exiting from the ion source and their motion in the x and y directions is controlled by the DC and RF potentials applied to the four rods. The motion of the stored ions in the quadrupole ion trap mass analyzer is controlled by applying potentials in three dimensions, x, y, and z. Due to the cylindrical QIT symmetry, the x and y dimensions can be combined in a radial coordinate r, where x 2 + y 2 = r 2 : q z = 2q r = 8zeV m (r +2z 2 )Ω 2 (Equation 2.12) a z = 16zeU m (r ₀ 2 +2z 2 )Ω 2 (Equation 2.13) 23
In the equations 2.12 and 2.13, ze is the charge of the ions, m is the ion mass, V is the amplitude of the RF potential (Ω), r0 is the inner radius of the ring electrode, and z0 is the distance from the center of the trap to an end cap (usually r0 = 2 z0 2 ). 1 To have stable trajectories, the ions should never reach or exceed the r0 and z0 coordinates. For a stable trajectory inside the trap, the qz of the ions should be less than 0.908 (Figure 2.10). The stability diagram (Figure 2.10) or Mathieu stability diagram is a two dimensional plot between q z and a z (Equation 2.12 and 2.13) showing the range of RF and DC potentials that lead to stable or unstable trajectories inside the field. Modern QITs are operated without DC potentials (az = ar = 0). For each trap, the frequency Ω is fixed and r0, z0, and e are constant values while qz depends on V and m/z. In Figure 2.10, the three heaviest ions (m1, m2, and m3) are trapped in the QIT because the value of q z is less than 0.908 while the lightest ion (m4) is ejected from the trap in the axial z direction because its q z value is more than 0.908. Ions with different m/z are successively ejected from the trap by slightly increasing their q z values to 0.908. After exiting the QIT, the 1, 45, 48 ions reach the detector. 24
Figure 2.10 Stability diagram for a QIT, in which four singly charged ions with the masses m4<m3<m2<m1 were injected. If no DC is used (U =0 and az =0), ion trajectories are determined only by the RF field (qz). Ions with qz<0.908 remain trapped (m1, m2, m3) and ions with qz >0.908 are ejected (m4) (reproduced with permission from reference 54). 2.3.4 Quadrupole/Time-of-Flight (Q/ToF) Mass Analyzer Numerous mass analyzers can be combined to enhance the performance of a mass spectrometer and overcome their individual weaknesses. These mass spectrometers are identified as hybrid mass analyzers, which include the quadrupole/time-of-flight (Q/ToF) mass spectrometer. The Q/ToF analyzer consists of a quadrupole and a time-of-flight analyzer located orthogonal to each other, with a collision cell placed between the two mass analyzers (Figure 2.11). 25
Figure 2.11 Schematic of the quadrupole/time-of-flight (Q/ToF) Mass analyzer (adapted with permission from reference 1). In MS experiments, only an RF potential is applied to the quadrupole mass analyzer. It acts as an ion guide that transmits all ions coming from the ion source to the time-of-flight tube. In MS/MS experiments, both RF and DC potentials are applied to the quadrupole mass analyzer so that only precursor ions of a specific m/z are allowed to pass through the quadrupole. Once the selected ions travel through quadrupole, they arrive in the collision cell where they collide with a neutral gas, usually argon (Ar), to form fragments. Then, the precursor ions and their fragments move to the time-of-flight tube, where they are separated according to their m/z value. Coupling a quadrupole with a time-of-flight analyzer in one mass spectrometer improves the sensitivity to attomole levels, the resolution to higher than 10000, the mass range to about m/z 20 000 and the mass accuracy to 5-10 ppm. 1 26
2.4 Detectors After the ions are generated in the ion source and separated in the mass analyzer, they reach the detector. The detector converts the ions into an electric current that is proportional to their abundance. Because only a small part of these ions reach the detector after leaving the mass analyzer at a specific time, the signal initially generated is low, so significant amplification is important to generate a measurable signal. 1 The microchannel plate (MCP) detector and Daly detector will be discussed as these were the detectors used in this dissertation. Figure 2.12 Schematic of a microchannel plate detector and the electron multiplication within the channels (adapted with a permission from reference 1). The microchannel plate (MCP) detector is a type of electron multiplier detector consisting of plate with a large number of parallel cylindrical channels inside. Each channel is a few millimeters long and few micrometers in diameter. All channels are covered by a semiconductor material to amplify the signal intensity by emitting electrons when struck with ion beams. The beams strike the detector wall at an angle so they can be 27
reflected and magnified. The resulting electron cascade is collected by using a metal anode at the output side and the current is measured (Figure 2.12). The input of the plate is kept at a negative potential of about 1 kv as compared to the output side. Figure 2.13 Schematic of a Daly detector (adapted with permission from reference 1). The Daly detector is a type of electro-optical ion detector that converts ions to electrons, and then to photons. This detector is composed of two conversion dynodes, a scintillator, and a photomultiplier tube (Figure 2.13). The ions coming from the analyzer will hit a conversion dynode. In the positive mode, positive ions will hit the negatively charged dynode, whereas in the negative mode, negative ions will hit the positive dynode. In both modes, secondary electrons will be generated and accelerated towards a phosphorescent screen and strike it to generate photons. The photons are detected and amplified by a photomultiplier. 1 28
2.5 Tandem Mass Spectrometry (MS/MS) Single stage mass spectrometry provides information about the mass of the molecular ion which is usually formed by a soft ionization method such as ESI or MALDI. In some cases, this information alone is not enough to determine molecular structure because the synthetic method was complex or the synthetic mechanism was unknown. In these instances, tandem mass spectrometry and multistage mass spectrometry methods, also known as two- or multi-dimensional mass spectrometry methods (MS/MS or MS n, respectively), can be performed to gain more information about the molecular structure under examination. 12 In MS/MS, two mass analysis events take place, which are separated either inspace or in-time (Figure 2.14). In-space tandem mass spectrometry employs two physically distinct mass analyzers, like the Q/ToF instrument. In-time tandem mass spectrometry involves events that take place sequentially in a single ion storage device, like the QIT instrument. For in-space tandem mass spectrometry, the first mass analyzer is utilized to select and isolate the precursor ion (or parent ion). This ion will be guided to a collision cell, which is located between the two analyzers, to be fragmented into product ions (fragment or daughter ions) by collisions with a neutral gas like argon or helium. The second mass analyzer will separate the fragment ions based on their massto-charge ratio (m/z). For in-time tandem mass spectrometry, the precursor ion is stored in the storage device while other ions are ejected. During a certain time period, the precursor ion is fragmented. These two steps of ejection and fragmentation can be repeated to form fragments of the fragments (MS n, where n is the number of generations of ions being analysed). 1, 45 29
Figure 2.14 Schematic of a tandem mass spectrometry in-space and in-time (adapted with permission from reference 1). In this dissertation, two methods were used to activate the precursor ion to cause fragmentation: collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). CAD is the most common gas phase fragmentation method in tandem mass spectrometry. It consists of a two-step process, which is collisional activation resulting in unimolecular dissociation. The precursor ion is accelerated to higher kinetic energy and then allowed to collide with a neutral gas such as helium or argon. During the collisions, some of the kinetic energy of the ion is converted to internal energy which causes some bonds to break and fragments the precursor ion into smaller pieces. 1 ETD is a relatively new technique, which causes fragmentation of multiply charged ions by electron transfer from ions of opposite charge in a gas phase ion-ion reaction that produces radical ions. The radical ions (charge-reduced precursor ions) dissociate without randomization of their internal energy, usually via radical-induced fragmentation. In contrast, CAD proceeds after randomization of the internal energy deposited in the collisions, leading to break up of the weakest bonds. 30
MS/MS in the MALDI-ToF/ToF instrument was done by using the LIFT technique. This instrument consists of a short linear ToF tube (ToF-1) coupled with a reflectron ToF analyzer (ToF-2). The LIFT (laser-induced fragmentation) device is located between ToF-1and ToF-2. An increased laser power is used to fragment the ions after they exit the MALDI source. A precursor ion and its fragment ions move together, as a family, because they have the same velocities. In ToF-1, a precursor ion and its fragment ions are selected using a timed ion gate because they have the same velocity as mentioned. The LIFT device is used to increase the kinetic energies of parent and fragment ions. They will gain different energies, depending on their mass, and that causes their velocities to change. The reflector ToF-2 will separate the fragment ions produced from the selected precursor according to their mass-to-charge ratio, as described in section 2.3.2. 59 2.6 Ion Mobility Mass Spectrometry (IM-MS) Traditional mass spectrometry can distinguish between ions according to their m/z values but it cannot separate them according to their architectures, conformations, or size. Ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) adds a new dimension in MS analysis. Ion mobility spectrometry measures the time it takes for an ion to drift through a buffer gas under a low electric field, while MS measures the corresponding mass/charge ratio. The IM-MS technique provides two dimensional separation, based on the m/z in the mass analyzer and drift time in the ion mobility chamber which depends on 3-D size and shape. By using IM-MS, ions that are isomers or 31
isobars, ions with the same m/z but different charge, and chemical noise become distinguishable if they travel through the ion mobility chamber in distinct times. The ions are separated there in time according to their shapes before mass analysis by the mass analyzer. There are four types of ion mobility spectrometry that can be interfaced with mass spectrometers: drift time, aspiration, differential, and traveling wave. 44, 60-62 Figure 2.15 Schematic of the Waters Synapt HDMS Q/ToF mass spectrometer 2006 Waters Corporation (adapted with permission from reference 62). The IM-MS work in this dissertation was performed on the Synapt HDMS Q/ToF mass spectrometer (Figure 2.15) which utilizes the traveling wave ion mobility spectrometry variant. The Synapt contains an ion mobility chamber which is located between the quadrupole and time-of-flight mass analyzers. The major components of the Synapt HDMS Q/ToF mass spectrometer include a traveling wave ion guide (TWIG) lens, a quadrupole, the triwave region with the IM chamber, and a time-of-flight tube. 32
The ions formed in the ion source travel through the ion guide where they are pulsed and move as a packet to the quadrupole. In MS mode, the quadrupole is used as an ion guide to move all ions to the triwave region. The triwave region is composed of three cells, which are the trap cell, ion mobility separation cell, and transfer cell (Figure 2.16). The triwave cells are composed of a series of ring electrodes (Figure 2.17) and focuses ions travelling axially through this region if opposite phases of an RF voltage are applied to adjacent rings. The trap and transfer cells are filled with Ar at 10-2 mbar, and the IMS cell is filled with N2 at ~ 1 mbar. Figure 2.16 Schematic of the triwave section of the Synapt HDMS system (adapted with permission from reference 44). The trap cell is utilized to collect ions as packets before they are sent to the ion mobility (IM) separation cell. Inside the IM cell, the ions are propelled forward by low voltage pulses (traveling waves) against the stream of a buffer gas, usually nitrogen, and in this process, they are separated according to their mobilities. The transfer cell is 33
utilized for passing the packets of mobility-separated ions to the ToF tube for mass analysis. In MS/MS mode, ion fragmentation can occur before IM separation in the trap cell or after IM separation in the transfer cell. MS/MS before the IM cell enables mobility separation of the fragment ions and MS/MS after the IM cell enables mobility 44, 61, 63 separation of the precursor ions subjected to MS/MS. Figure 2.17 Schematic of a stacked ring ion guide (adapted with permission from reference 44). Adjacent rings in the triwave area are connected to opposite phases of an RF voltage, which provides axial movement to the entering ions. In the IM cell, low pressure nitrogen gas flows against the entering ions, slowing them down or preventing their axial movement. A transient DC voltage is applied successively to adjacent ring electrodes in a repeating sequence at regular time intervals to drive the ions through the gas (Figure 2.18). The traveling waves created this way drive the ions through the IM cell where they are separated. Traveling waves with heights up to 25 V and velocities in the range between 300 600 m/s can be employed in the Synapt HDMS instrument. Weak traveling 34
waves with typical height and velocities of 1-2 V and 300 m/s, respectively, are maintained in the transfer cell to preserve the ion mobility separation of the ions until they reach the ToF mass analyzer. There are usually no waves in the trap cell. The larger ions will move more slowly in the IM cell because they undergo more collisions with neutral molecules and thus stay longer behind the waves while the smaller ions will move faster. Ions with higher molecular weight can have short drift times if they also have 44, 61, 63 higher charge states. Figure 2.18 Schematic of the operation of a traveling wave ion guide containing ring electrodes for transferring the ions through the buffer gas (adapted with permission from reference 63). Traditional drift ion mobility spectrometers measure the time that ions take to move through the buffer gas when a low static field is applied to the cell. Under these conditions, the velocity of the ions (υ D ) is proportional to the electric field (E), as shown in Equation (2.14), and the drift time (t D ) taken to traverse the length (L) of the drift cell is shown in Equation (2.15). The proportionality constant (K) in these equations is named 35
the ion mobility constant and is directly proportional to the ion cross-section (Ω) as shown in Equation (2.16). υ D = KE (Equation 2.14) L t D = KE (Equation 2.15) K = 18π 16 ze [ 1 + 1 ] 1 k b T m 1 m N N 1 Ω (Equation 2.16) In the latter equation, N is the number density (pressure) of the buffer gas, T is the effective temperature, k b is the Boltzmann constant, m 1 is the mass of the ion, m N is the mass of the buffer gas atom or molecule, z is the ion charge, e is elementary charge and Ω is the collision cross-section of the ion. 64 The cross-sections of the ions provide detailed information about their structures, geometries and sizes. The collision cross-section (CCS) of an ion can be determined directly from equation (2.17) by using a standard drift tube separator with constant electric field. 65 In cases where direct measurement of the neutral gas purity, pressure and temperature is difficult and the electric field inside the ion mobility region is not constant (as in the triwave IMS cell), the drift time scale must be calibrated using the drift times of ions with known collision cross-sections. When the collision cross-section is known and a traveling wave field is used, Equation 2.17 becomes Equation 2.18, which contains parameters A and B: A is a parameter for the electric field and B is a correction factor for the non-linearity of the triwave device. Ω= 18π 16 Ze k b T [ 1 + 1 ] t D E m 1 m N L 760 T 1 P 273.2 N (Equation 2.17) 36
Ω= 18π 16 ze [ 1 + 1 ] 760 K b m 1 m N P T 273.2 1 N A t D B (Equation 2.18) According to this equation, the CCS depends on the masses of the ions and their charges. Dividing Equation 2.18 by ze and [ 1 + 1 ], gives Equation 2.19 and a normalized m 1 m N CCS (Ω`) which is independent of masses and charges. The parameters in Equation 2.19, including the constant A, can be combined in a single constant A`, which can be viewed as the correction factor for the temperature, pressure and electric field parameters (Equation 2.20). The relationship between CCS and normalized CCS is shown in Equations 2.21 and 2.22. 64-65 Ω`= 18π 16 1 k b T 760 P T 273.2 1 N A t D B (Equation 2.19) Ω`=A` t D B (Equation 2.20) Ω= ze [ 1 m 1 + 1 m N ] Ω` (Equation 2.21) Ω= ze [ 1 m 1 + 1 m N ] A` t D B (Equation 2.22) t D` = t D - [ c m z ] (Equation 2.23) 1000 The drift time correlated to Ω` should be corrected to the time spent in the IM cell, but the experimental drift time from the Synapt is the time spent in the IM cell and in the ToF analyzer. The corrected (IMS) drift time (t D` ) is obtained by Equation 2.23. In this equation, c is the enhanced duty cycle (EDC) delay coefficient. It is dependent on the 37
specific instrument and its value is between 1.4-1.6. To derive the experimental CCS for an ion with the Synapt instrument, ions of different proteins like cytochrome c, equine myoglobin, bovine ubiquitin, and insulin, and/or ions from polymers like polyalanine can be used as standards to calibrate the Triwave drift time scale. The standards and the ions of unknown CCS are run under the same instrument conditions. 65, 66 2.7. Liquid Chromatography Mass Spectrometry (LC-MS) Liquid chromatography (LC) was developed in 1969 as a powerful analytical tool, and since that time, it continues to be one of the most efficient separation methods for the analysis and separation of a wide range of nonvolatile mixtures into their components based on their physicochemical or chemical interactions with two phases. To analyze a complex mixture by HPLC, the sample must be dissolved in a solvent before it is introduced into the instrument. The mobile phase, which can be one or more liquid solvents, passes through the column, which is packed with a stationary phase. The components move with different rates of migration depending on their interactions with both phases, so they elute from the column at different times into the detector. There are various detectors that can be coupled to LC, but mass spectrometry hyphenated with LC combines the strength of the LC and MS techniques for both qualitative and quantitative 67, 68 analysis of the components in complex mixtures. There are several classes of HPLC depending on the physical nature of the stationary phase: normal phase, reverse phase, ion exchange, size exclusion, affinity, and chiral chromatography. In normal phase HPLC, the stationary phase is polar which can contain nitro or amino groups, while the mobile phase consists of non-polar solvents. The 38
most polar compound in the analyte sample has the longest retention time because it interacts most strongly with the stationary phase, thus staying for a long time inside the column; the least polar component elutes out first. In reverse phase HPLC, the stationary phase is non-polar, usually containing hydrophobic substituents (alkyl chains), while the mobile phase consists of polar solvents. In this case, the least polar compounds have the longest retention time and the most polar component elutes out first. Normal-phase (NP) and reverse-phase (RP) HPLC are the most widely used LC methods. In the ion exchange HPLC, the stationary phase is a high-mass polymer carrying a positive or negative charge whereas the mobile phase is a buffer and the ph increases during the elution step. In sizeexclusion chromatography, the stationary phase is silica or polymer particles with a network of uniform pores, while the mobile phase and the eluent are only solvent. Components in the sample separate in this chromatography by their shape or size; larger components spend less time inside the pores and, hence, move faster and elute out first. 68, 69 In affinity HPLC, a guest ligand, such as a virus, enzyme or protein, is bonded to the host ligand on the packing particles, such as an antibody or enzyme, by biospecific interactions. In chiral chromatography, the stationary phase contains chiral molecules immobilized on the packing particles, and the enantiomeric component is bonded to the chiral stationary phase by stereoselective binding. 22 A LC-MS system consists of six components; the mobile phase supply system, pump system, sample injector, column, MS detector, and data processing system (Figure 2.19). The mobile phase supply system contains multiple mobile phase reservoirs, and the mobile phase must be pure and of special chromatography grade. The pump is employed to help carry the analytes through the column and deliver the mobile phase through the 39
column with a reasonable flow rate. 68, 70, 71 The most important part in the HPLC system is the column, where the separation takes place. It is a stainless steel tube packed with the stationary phase. After the components of a mixture have been separated, each analyte leaves the column and moves into the MS detector. Modern LC systems are interfaced with an ESI or APCI for analyte ionization. 71 Figure 2.19 Schematic of the basic components of an HPLC-MS system (adapted with permission from reference 71). HPLC is time consuming, requires large volumes of organic solvent, and may have low resolution and sensitivity. To overcome these problems, ultra-performance liquid chromatography (UPLC) was developed in 2004. The UPLC column contains smaller size particles, usually 1.7 µm, and is operated at higher flow rates, which help to 21, 69- decrease the run time as well as enhance the sensitivity and efficiency of separation. 77 In this dissertation, ESI was interfaced with a RP-UPLC system to characterize alkyl glycoside surfactants. Most LC applications use reverse-phase liquid chromatography (RP-HPLC) because water or aqueous mixtures are utilized as the mobile phase. The most widely 40
utilized stationary phases contain organic nonpolar functional groups such as alkyl (octyl (C8), octadecyl (C18)) or aryl phenyl groups, which are bonded to silica by reacting its hydrolyzed surface Si-OH groups with an organochlorosilane Cl-Si-R to make Si-O-Si-R (R=alkyl or phenyl) linkages. 78 In RPLC, the least polar components from the analyte sample are adsorbed strongly on the hydrophobic groups (R) of the stationary phase, while the most polar components travel faster with the polar mobile phase, which is an aqueous mixture of organic solvents, and elute first. Mobile phase solvents generally used in RPLC are water, acetonitrile, methanol and isopropanol. 78 In chromatography, the partition (distribution) equilibrium, which describes the interaction of an analyte component with the stationary vs. the mobile phase, controls the separation. Components similar with the mobile phase will move through the column faster and elute earlier than components that interact with the stationary phase. This distribution coefficient or distribution constant (K) is equal to the ratio between the analyte concentrations in the stationary (c S ) and mobile phases (c M ), as shown in Equation 2.24. K = c S c M (Equation 2.24) Chromatographic data are presented in the chromatogram, which is plotted with time or the volume of mobile phase on the x-axis and the signal or detector response on the y-axis. A chromatographic peak is identified by the retention time t R and the width of the peak at half height W1 2. The column efficiency describes how well the sample is separated as it moves through the column. It is measured by the number of theoretical plates (N) (Equation 2.25) or by the height equivalent of a theoretical plate (H) (Equation 41
2.26), where L is the length of the column packing. By increasing the number of the theoretical plates in a column and decreasing the height of the plate, column efficiency is increased and this leads to narrower and better resolved peaks in the chromatogram. N = 5.54 ( t R W 1 2 ) 2 (Equation 2.25) H = L N (Equation 2.26) H = A + B + CU (Equation 2.27) U In 1956, Van Deemter introduced the first equation to explain how experimental factors, such as the diameter of packing particles and velocity of the mobile phase, can affect plate heights and how that can cause band broadening and decrease column efficiency. The Van Deemter equation contains three factors, which are eddy diffusion, longitudinal diffusion, and mass transfer resistance. By controlling these factors, the distribution of the analyte components through the column can be controlled to minimize band broadening. In Equation 2.27, H is the plate height, A describes multiple path effects or eddy diffusion, B is the longitudinal diffusion coefficient, C is mass transfer coefficient for a stationary liquid phase and U is the linear velocity of the mobile phase. Eddy diffusion is known as the multipath effect (A Term) which arises from the fact that the analyte molecules move randomly, taking different paths through the stationary phase; since the flow rate of the mobile phase is constant, some analyte molecules will travel short distances and elute early, while others will follow longer paths until they elute. This effect causes band broadening and poor resolution. These differences in paths can be caused by imperfect column packing, in which the shapes or sizes of the packing 42
particles are different. This factor can be minimized by using a column packed with particles with the same shape and small size. Operating Curve Theroetical Plate Height (H) Mass Transfer (Term C) Eddy Diffusion (Term A) Longitudinal Diffusion (Term B) Mobile Phase Linear Velocity (U) Figure 2.20 Van Deemter plot (dashed line) and individual plots of the terms of the Van Deemter equation. Longitudinal diffusion is also known as flow distribution (B Term). Diffusion results from migrations of the analyte components, while they are flowing down the column from a more concentrated region into a more dilute region. This diffusion effect increases as the time spent in the column increases. This factor can be minimized by increasing the flow rate of the mobile phase. Mass transfer diffusion is also known as resistance to mass transfer (C Term). It explains the relationship between the time it takes for an analyte component to equilibrate between the two phases and the mobile phase flow rate. If the time spent in the stationary phase is longer or shorter than the time required for the 43
partition equilibration to take place, band broadening can occur. To decrease this effect, small diameter and porous stationary phase particles can be used. Both the B and C terms depend on the flow rate of the mobile phase, which can be optimized to minimize the effect of these terms. The Van Deemter plot (Figure 2.20) is a plot of the mobile phase linear velocity (U) as the x-axis and the theoretical plate height (H) as the y-axis. The plot explains how the B and C terms are affected by changing the mobile phase flow rate. The optimum flow rate, which can be obtained from the plot, 68, 70, 78 reduces the dispersion (band broadening) and increases the separation efficacy. 44
CHAPTER III MATERIALS AND INSTRUMENTATION 3.1 Materials Methanol, water, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, and formic acid, all of HPLC grade, were purchased from Sigma-Aldrich (St. Louis, MO). Ammonium acetate in LC-MS grade was received from Fisher Scientific (Pittsburgh, PA). Sodium trifloroacetate (NaTFA), potassium trifloroacetate (KTFA), lithium trifloroacetate (LiTFA) and silver trifloroacetate (AgTFA), used to improve ionization of the samples, were obtained from Fluka (Buchs, Switzerland). α-cyano-4- hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid, SA), trans-2-(3-(4-tert-butylphenyl)-2-methyl-2-propenyliedene)malononitrile (DCTB), and 1,8-dihydroxy-9,10-dihydroanthracen-9-one (dithranol, DIT) were the MALDI matrices used to analyze the samples in this dissertation. CHCA and SA were purchased from Sigma Aldrich (St. Louis, MO). DCTB was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and dithranol was purchased from Alfa Aesar (Ward Hill, MA). The peptide (VPGVG)₂, polyacrylic acid (PAA) and two hybrid materials (PtBA- VG2) and (PAA-VG₂) were received from Dr. Xinqiao Jia s group (University of 45
Delaware). N-octyl-β-D-glucoside (β-c₈g₁) and n-dodecyl-α-d-maltoside (α- C12G2)[high alpha isomer] were obtained from Affyymetrix, Inc (Maumee, OH) and were of Analytical grade quality. These surfactants were used to dissolve the VG2 peptide. HA peptide, BMP-2 peptide, and BMP-2-(HA)2 hybrid were received from Dr. Matthew Becker s group (University of Akron). Alkyl polyglycoside samples, viz. Plantacare 818, Plantaren 1200 and Plantaren 2000, were received from BASF corporation (Wyandotte, MI). All materials were used in the condition received from their supplier without purification. 3.2 Instrumentation The following sections describe the running conditions and instrument settings to obtain the data in this dissertation. Figure 3.1 Schematic view of the Bruker HCT ultra II ESI-QIT mass spectrometer (adapted with permission from reference 48). 46
3.2.1 HCT Ultra II ESI-QIT Mass Spectrometer Some of the experiments reported to characterize the peptides and surfactants discussed in chapter IV,V and VI were performed using a HCT Ultra II quadrupole ion trap (QIT) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with an electrospray ionization (ESI) source and a negative chemical ionization (nci) source for electron transfer dissociation studies. A scheme of this instrument is shown in Figure 3.1 and such a QIT with an external nci source for ETD experiments is shown in Figure 3.2. The liquid solution of the samples is introduced by direct injection to the electrospray chamber through a fine metal needle at a flow rate of 180 μl/h, which is controlled by using a syringe pump. The grounded needle is surrounded by nebulizer gas, which is nitrogen gas at a pressure adjusted from 0-80 psi. It flows through the nebulizer and joins the sample solution at the needle tip. The pressure used in this dissertation was 10 psi. In the spray chamber, a heated drying gas (nitrogen) is used to help evaporate the solvent from the droplets. The drying gas temperature can be varied between 35-365 ºC at a flow rate of 0-12 L/min. In this dissertation, the optimized settings were 8 L/min at 300 ºC. Figure 3.2 QIT with external nci source for ETD experiments in the HCTultra II QIT mass spectrometer (adapted with permission from reference 48). 47
The analyte solution is sprayed into the spray chamber, which is operated at atmospheric pressure. Droplets are produced at the needle tip by an electrostatic gradient that is created by the voltage difference between the spraying needle and the entrance of the glass capillary that guides the ions into the QIT. In positive mode, the entrance of the glass capillary is held between -4.5 kv and -1.5 kv relative to the spraying needle, and at -0.5 kv relative to the end plate. The heated drying gas helps to evaporate the solvent from the droplets. The electrostatic gradient also ensures movement of the charged droplets towards the glass capillary. The glass capillary is an ion transfer device and transmission region that transfers the ions from atmospheric pressure to vacuum. The ions exiting the capillary are pushed through the skimmer and focused by two octapoles and two lenses in the direction of the ion trap. The drying gas and solvent molecules are removed by the skimmer device. The voltages of skimmers, octapoles and exit lenses are set at certain values to carry all the ions produced by ESI to the ion trap and decrease interference of background noise. The operation principle of a QIT was discussed in section 2.3.3. Ions ejected from the ion trap hit a Daly Detector to produce an electrical current that is proportional to the abundances of the ions. 48 The operation principle of a Daly detector was discussed in section 2.4. All ESI spectra in Chapters IV and VI were measured in positive mode. 3.2.2 Ultraflex III ToF/ToF Mass Spectrometer The studies performed in chapters IV, V and VI utilized an Ultraflex III ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA). It consists of two time-of-flight analyzers and is equipped with a MALDI source operating with an Nd: YAG laser at 355 48
nm. The first time-of-flight tube (ToF-1) is a short linear tube and the second time-offlight tube (ToF-2) is a reflectron analyzer and is interfaced with the first mass analyzer. In single stage mass spectrometry mode, the two ToF analyzers work as one combined linear (linear mode) or reflectron (reflectron mode) ToF analyzer. In tandem mass mode, the two ToF analyzers operate separately by use of the LIFT mode as described in section 2.5. All samples were prepared by using the sandwich method. In this method, a solution of matrix with or without cationizing agent is applied on the plate, then the sample is applied onto the dried matrix spot, and then a second layer of matrix with or without salt is applied again on the top of the dried sample. MALDI spectra in Chapters IV, V and VI were measured in positive or negative reflectron mode. The instrument was calibrated for each measurement using poly(methyl methacrylate) (PMMA); Mn=2000 as a positive ion external standard (Sigma-Aldarich, St.Louis, MO) or polystyrene (PS); Mn=550 as a positive ion external standard (Scientific polymer, Ontario, NY) for low molecular mass samples and polystyrene sulfonate (PS- SO3H); Mn=2500 as a negative ion external standard. The operation principle of MALDI and the ToF/ToF mass spectrometer was previously described in section 2.2.2 and section 2.3.3, respectively. 3.2.3 Synapt HDMS Ion Mobility Mass Spectrometer The studies performed in chapters IV, V and VII utilized a Synapt HDMS TM mass spectrometer (Waters Corporation, Milford, MA) equipped with an electrospray ionization source, a quadrupole orthogonal acceleration time-of-flight mass analyzing 49
system as well as a tri-wave ion mobility region (Figure 2.15). The operation principle of this instrument and the triwave ion mobility function were explained in section 2.6. 3.2.4 Acquity UPLC The reverse-phase liquid chromatography (RP-LC) study reported in Chapter VI was carried out using an Acquity UPLC TM system (Figure 3.3) supplied by Waters Corporation (Milford, MA). The Acquity UPLC TM system consists of a binary pump, autosampler, column heater, and degasser. It was coupled to the Synapt HDMS Q/ToF mass spectrometer. The pumps in the UPLC system can handle pressures up to 15,000 psi and the particle size in the UPLC columns is 1.7 μm, resulting in narrow peaks, high resolution, and shorter elution times as compared to traditional HPLC. 79 Figure 3.3 Schematic view of the Acquity UPLC system 2004 Waters Corporation (adapted with permission from reference 79) 50
CHAPTER IV TOP-DOWN MASS SPECTROMETRY OF HYBRID MATERIALS WITH HYDROPHOBIC PEPTIDE AND HYDROPHILIC POLYMER BLOCKS 4.1 Background Coupling peptides, which can be poly(amino acids) or designed peptide sequences, 80 with synthetic polymers to make new materials is an emerging research area that has been developed in the last few years. These materials are known by different names, like polymer-peptide conjugates, hybrid materials and bioactive materials. 81 In the past years, many peptides have been approved as drugs because of several useful properties, including their ability to self-assemble into precisely defined structures and respond to external stimuli. In addition, peptide drugs with diverse conformations and functionalities can be prepared that show remarkable selectivity and specificity in their interactions with their targets. 82 On the other hand, using peptides in biomedical research is still limited because of their physical properties, including sensitivity to temperature, ph, organic solvents, and susceptibility to degradation. Attaching a synthetic polymer to these materials helps to control their physical and chemical properties, such as viscosity and responsiveness to external stimuli (smart behavior). 51
The new hybrid materials (polymer-peptide conjugates) have high complexity and high modularity, as they combine natural and synthetic properties, 83, 84 and they can be used in biological and non-biological applications. 84 The properties of such hybrid materials depend on the sequence of amino acids and choice of synthetic polymers, as well as on the methods used for the synthesis of both these compounds. 83 The most common methods for coupling a peptide and a polymer are the succinimide, Schiff base, click chemistry, and thiolmaleimide methods. A widely used click chemistry reaction is the azide-alkyne Huisgen cycloaddition reaction, in which terminal azide groups react with terminal alkyne groups in the presence of a copper catalyst to produce triazole rings via 1, 3-dipolar cycloaddition. 85-90 The advantages of this reaction are high efficiency, high selectivity, ease of product purification, and compatibility with a wide diversity of functional groups. Many synthetic polymers have been successfully joined to peptides, including poly(ethylene glycol), 92-100 polystyrene 101-104 and poly(butyl acrylate). 105-109 Polypeptides derived from Elastin are one class of synthetic peptides that have been used widely in drug delivery 110 and tissue engineering, either by themselves 111 or conjugated with a polymer. 112 Elastin is a protein that affords flexibility to many connective tissues, blood vessels, lungs, and the skin of vertebrates. 113-116 This protein has two alternating domains. The first domain is rich in hydrophobic amino acids (Gly, Ala, Val, and Pro) and is the peptide sequence which gives flexibility to the tissues. The second domain is hydrophilic, enriched with Ala and Lys, and makes cross-links to other polypeptide chains with its Lys residues. 114, 117 The structure of the polypeptide (VPGVG)n is 83, 91 52
changed from hydrophilic random coil to hydrophobic β-spiral when the temperature is increased because of inter- and intramolecular hydrogen bonding interactions, which cause the aggregation and precipitation of the polymer. Changes in polypeptide (VPGVG)n affect any polymer coupled with it, even with only one repeat unit of the polypeptide. 118-120 Different analytical techniques like Differential Scanning Calorimetry (DSC), Pressure Perturbation Calorimetry (PPC), Circular Dichroism Spectroscopy (CD), Ultraviolet (UV) absorption, 115 Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy have been used to study conformational changes in different sequences of GVGVP. 115,120,121 Peptide-polymer conjugates is a fast growing field but the characterization of such products is very difficult because their solubility limits the analytical techniques that are suitable for the examination of their structures. 89 Literature about the characterization of hybrid materials is scarce. Polystyrene-peptide conjugates have been analyzed by UV spectrometry, gel permeation chromatography (GPC), Proton Nuclear Magnetic Resonance ( 1 H NMR), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). 102 Poly(ethylene glycol) (PEG)-peptide conjugates have been analyzed by 1 H NMR, GPC and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS). 122 Different poly(ethylene glycol) (PEG)-peptide conjugates have been analyzed by 1 H NMR, reverse-phase HPLC fractionation and MALDI-ToF mass spectrometry identification of the collected fractions. 123-124 Butyl acrylate peptide conjugates have been analyzed by 1 H NMR spectroscopy and size exclusion chromatography (SEC). 125 53
Mass spectrometry is introduced here as a comprehensive characterization technique for hybrid materials that are difficult to solubilize and/or purity. This chapter describes new top-down mass spectrometry methods for the analysis of hybrid materials containing hydrophilic poly(t-butyl acrylate) (PtBA) or polyacrylic acid (PAA) blocks attached to blocks of the hydrophobic peptide (elastin mimic) VPGVGVPGVG (VG2). The VG2 peptide and polyacrylic acid are also characterized individually. The methods used include MALDI-ToF-MS and MS 2, ESI-QIT-MS, and ESI-Q/ToF-MS coupled with ion mobility separation. In addition, molecular modeling is used to calculate the structures and collision cross sections of the hybrid materials for comparison with experimental results obtained by IM-MS experiments. 4.2 Sample Preparation and Instruments Used The samples analyzed were prepared in Dr. Jia s group (University of Delaware) and include the peptide (VPGVG)₂, polyacrylic acid (PAA) and two hybrid materials, (PtBA-VG2) and (PAA-VG₂). 126,127 4.2.1 Sample Preparation for MALDI-ToF/ToF-MS MS and MS² experiments were performed on a Bruker UltraFlex III MALDI tandem time-of-flight (ToF/ToF) mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a Nd:YAG laser emitting at 355 nm. The instrument was operated in positive or negative ion mode. The VG2 peptide was analyzed using the sandwich method and α-cyano-4- hydroxycinnamic acid (CHCA) as matrix. A solution of CHCA matrix was prepared in 54
THF at the concentration of 20 mg/ml. A solution of sodium trifluorocactate (NaTFA) salt was prepared in THF at the concentration of 10 mg/ml. Matrix and salt solutions were mixed in the ratio 100:10 (v/v %). A solution of the sample was prepared in DMSO at the concentration of 10 mg/ml. This sample preparation procedure led to the formation of [VG2 + Na]+ ions. For solvent-free analysis, CHCA and the VG2 peptide were mixed and grinded without adding any solvent. The mixed powder was transferred to the MALDI sample target. This sample preparation procedure led to the formation of [VG₂ + Na]+ ions. For polyacrylic acid (PAA), CHCA or sinapinic acid (SA) served as matrix in positive and negative ion mode, respectively. Solutions of the CHCA matrix (in THF) or the SA matrix (in ACN:H2O 70:30, v/v %) were prepared at 20 mg/ml. A solution of NaTFA salt was prepared in THF at 10 mg/ml. Matrix and salt solutions were mixed in the ratio 100:10 (v/v %). A polyacrylic acid solution (10 mg/ml) was prepared in THF or THF: MeOH (50:50, v/v %). Matrix/salt and sample were applied onto the MALDI target plate by the sandwich method. This sample preparation protocol led to the formation of [M + Na]+ ions in positive and [M - H] ions in negative mode. For poly(t-butyl acrylate) block VG2 (PtBA-VG2) hybrid materials, DCTB, viz. {trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenyldene] malononitrile}, served as matrix in positive ion mode. A solution of the matrix (20 mg/ml) was prepared in THF, and a solution of potassium trifluorocactate (KTFA) salt (10 mg/ml) was prepared in THF. Solutions of the matrix and salt were mixed in the ratio 100:10 (v/v %) and the resulting mixture was used for the top and bottom layers of the sandwich deposition method. A solution of PtBA-VG2 hybrid (10 mg/ml) was prepared in a 25:75 (v/v %) 55
mixture of ammonium acetate buffer (ph=6.64) and MeOH and was used for deposition of the middle layer. This sample preparation protocol led to the formation of [M + K]+ ions. For polyacrylic acid block VG2 (PAA-VG2) hybrid materials, CHCA served as matrix in positive and negative ion modes. A solution of the matrix (20 mg/ml) was prepared in THF, and a solution of PAA-VG2 (20 mg/ml) was prepared in DMSO (99.9 %; Aldrich). The sandwich method was used. This sample preparation protocol led to the formation of [M + Na]+ ions in positive and [M - H] ions in negative mode. 4.2.2 Sample Preparation for ESI-QIT-MS MS and MS² experiments were performed on a quadrupole ion trap (QIT) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with an electrospray ionization (ESI) source operated in positive ion mode. VG2 sample solutions were injected into the instrument by direct infusion at a flow rate of 3 μl/min. The entrance of the vacuum system held at -3.5 kv, the nebulizer gas pressure at 10 psi, and the drying gas flow rate and temperature at 8 L/min and 300 C, respectively. The VG2 peptide was dissolved in 56 mm aqueous ammonium acetate buffer (ph=6.64) at the concentration of 0.01 or 0.05 mg/ml and 10 % (v %) MeOH was added to help buffer evaporation and spray formation. CAD experiments were carried out by using helium as collision gas and setting the excitation amplitude value between 0.18 and 0.80 (arbitrary units), depending on the precursor ion isolated. For the experiments involving ETD, the anion reagent species were generated in a negative chemical ionization (nci) source, which was tuned to 56
maximize the generation and transmission of the ETD reagent ions (fluoranthene radical anions) as follows: reagent ion ICC 100000, ionization energy 70 ev, emission current 2.0 μa, reactant remove cut off m/z 210 and methane as buffer gas. After the accumulation of both precursor ions and ETD reagent radical anions inside the ion trap, the reaction time was set in the range between 400-750 ms, depending on the peptide investigated and the precursor ion selected. 4.2.3 Sample Preparation for ESI-Q/ToF-MS MS and MS² experiments were performed on a Waters Synapt HDMS quadrupole/time-of-flight (Q/ToF) mass spectrometer (Waters, Beverly, MA, USA), equipped with an electrospray ionization (ESI) source operated in positive or negative ion mode. The sample solution was introduced to the ESI source by direct infusion. The instrument was operated in positive ion mode with a capillary voltage of 3.15 kv, cone voltage of 35 V, sampling cone voltage of 3.2 V, source temperature of 80 C, and desolvation temperature of 150º C. The instrument was operated in negative ion mode with a capillary voltage of 1.66 kv, cone voltage of 51 V, sampling cone voltage of 1.1 V, source temperature of 80 C, and desolvation temperature of 150º C. The VG2, the sample was dissolved in ammonium acetate buffer (ph=6.64) at 0.01 or 0.05 mg/ml and 10 % (v %) of MeOH was added to help buffer evaporation. The instrument was operated in positive ion mode. The polyacrylic acid (PAA) sample was dissolved in THF: MeOH (50:50, v: v %) at 0.1 mg/ml with 1% (v %) of formic acid. The instrument was operated in positive ion and negative ion modes. The PtBA-VG2 sample was dissolved in a 50:50 (v/v %) mixture of ammonium acetate buffer (ph=6.64) 57
and MeOH at the concentration of 0.1 mg/ml and 1% (v %) NaTFA salt, KTFA salt or formic acid was added to this solution. The instrument was operated in positive ion mode. The PAA-VG2 sample was dissolved in ammonium acetate buffer (ph=6.64) at 0.01 mg/ml and 10 % (v %) MeOH was added to help the buffer evaporate. The instrument was operated in positive ion and negative ion modes. Ion mobility separation was achieved by tuning the wave height and wave velocity to 12 V and 350 m/s (for VG2), and 8 V and 350 m/s (for PAA and hybrid materials), respectively; the wave height and the wave velocity in the trap cell were set to 0.5 V and 300 m/s, respectively; the wave height and wave velocity in the transfer cell were set to 0.2 V and 248 m/s, respectively. The nitrogen gas (drift gas) flow rate was 22 L/h. Tandem mass spectrometry experiments were performed in the transfer cell, located after the ion mobility chamber, using argon as collision gas. 4.2.4 VG2 Peptide with Surfactant N-dodecyl-α-D-maltoside (α-c12g2) [high alpha isomer] was obtained from Affymetrix, Inc (Maumee, OH) and was of analytical grade quality. It was dissolved in H2O at the concentration of 0.01 mg/ml. The peptide was subscquenray dissolved in this solution at the concentration of 0.01 mg/ml or 0.05 mg/ml. The final sample solution was introduced by direct infusion to the ESI source of the quadrupole ion trap (QIT) mass spectrometer (Bruker Daltonics, Billerica, MA) and the Synapt HDMS quadrupole/timeof-flight (Q/ToF) mass spectrometer (Waters, Beverly, MA, USA). The instruments were operated in positive ion mode. 58
4.3 Characterization of VG2 Peptide Peptide analysis by mass spectrometry can provide information about the molecular mass, sequence, terminal end groups 1 and shape of a peptide. 128, 129 Sequences are generally identified by the fragments formed in tandem mass spectrometry experiments. Peptide fragments are named according to the Biemann nomenclature, shown in Figure 4.1. Fragments containing the N-terminal side are named, a n, b n and c n, and fragments containing the C-terminal side are named, y n, x n and z n where n is the number of amino acid residues in the fragment. 1 Figure 4. 1 Nomenclature of peptide fragment ions according to Biemann. This chapter reports the procedures developed for the analysis of the VG2 peptide. VG2 is a hydrophobic peptide with ten amino acid residues. These include four glycine units (G = C₂H₃O₁N₁ = 57.021449 Da), four valine units (V = C₆H₉O₁N₁ = 99.068399 Da) and two proline units (P = C₆H₇O₁N₁ = 97.052749 Da). Based on the synthetic route used to prepare VG2, 127 its sequence should be XVPGVGVPGVGX where X is acetylated propargyl glycine (C₇N₁H₈O₂ =138.0555 Da) at the N-terminus and amidated propargyl 59
(A) [VG₂+Na] + 1090.01 incomplete sequences 400 500 600 700 800 900 1000 m/z (B) [VG₂+Na] + 1090.67 1030 1040 1050 1060 1080 1100 1200 1300 m/z Figure 4.2 A) MALDI-MS spectrum of VG2 peptide acquired using the sandwich method. The peptide was dissolved in DMSO at 20 mg/ml. CHCA (20 mg/ml) matrix was mixed with NaTFA salt in the ratio (100:10, v/v %). B) MALDI-MS spectrum of VG2 peptide mixed with CHCA without using solvents. 60
glycine (C₅N₂H₇O =111.05584 Da) at the C-terminus. The calculated mass of the protonated peptide is 1068.584055 Da using the monoisotopic masses of the elements in the peptide. The peptide is hydrophobic and could only be dissolved in DMSO and ammonium acetate buffer with a 6.64 ph. Figure 4.3 Structures of XVPGVGVPGVGX and XGVGPVGVGPVX. 4.3.1 Characterization of VG2 by MALDI-ToF/ToF-MS and MS 2 Figure 4.2A shows the MALDI-MS spectrum of VG2 acquired using a DMSO solution of this peptide, and Figure 4.2B shows the MALDI-MS spectrum of VG2 acquired using the solvent-free method. In both of these experiments, CHCA was the matrix, but SA matrix gave the same results. The most abundant peaks in Figure 4.1 A and B arise from the quasi-molecular ion at m/z 1090, i.e. [VG2+Na]+; the other ions originate from incomplete sequences. The desired sequence XVPGVGVPGVGX and the 61
192.7 290.7 347.7 403.7 756.9 444.7 483.8 543.8 699.8 793.9 600.8 953.0 893.0 reverse sequence XGVGPVGVGPVX are isomers with equal molecular weights (Figure 4.3). Tandem mass spectrometry can help to identify the correct VG2 peptide sequence. y 10 y 8 y 7 y 6 y 5 y 4 y 9 a ₂ a ₄ a ₆ a ₉ a ₆ 639.8 y 9 853.9 a₉ [XVG₂X + Na] + 1090.0 y 4 a₄ y 5 y 1 y 2 y 3 y 6 y 7 y 8 a ₈ y 10 100 200 300 400 500 600 700 800 900 1000 1100 m/z Figure 4.4 MALDI-MS 2 spectrum of sodium cationized VG2 peptide (m/z 1090.0) acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum. X designates the propargyl-substituted end groups of the peptide (138 Da at the N-terminus and 111 Da at the C-terminus), which allow it to react with azide-terminated polymer to form a peptide-polymer hybrid material. The MALDI-MS 2 spectrum of the [VG2+Na]+ ion (m/z 1090.01) is shown in Figure 4.4. It agrees well with sequence XVPGVGVPGVGX, comprising an acetylated propargyl glycine at the N-terminus and an amidated propargyl glycine at the C-terminus. The most abundant fragments are series a n, generated by cleavages at the C α -C(=O) bonds, and series y n, generated by cleavages at the C(=O)-NH bonds. The intensity of fragments y 9 and a 6 is high because of the favored cleavage of C(C=O)-NH bonds at the 62
N-terminal side of proline, which is the amino acid unit with the highest proton affinity in the VG2 peptide. 130-132 The proton affinities for proline, valine and glycine are 222.0, 216.5 and 210.5 kcal mol -1, respectively. 132 The presence of a complete yn series from y10 to y1, and of intense a n ions with longer chains (a 6, a 9 ) suggest a preference for sodium ion to attach at the C-terminal end group and near the other C-terminal residues. Figure 4.5 MALDI-MS spectrum of XVG2 acquired using a 20 mg/ml DMSO solution of the peptide and the sandwich method. CHCA (20 mg/ml) matrix mixed with NaTFA salt in the ratio (100:10, v/v %) was used for the bottom and top layers. To support the conclusion that Na + is bonded near the C-terminal end group of XVG2X, the peptide XVG2 was also analyzed. XVG2 had the same sequence as XVG2X and an acetylated propargyl glycine at the N-terminus, but the C terminal propargyl glycine residue was missing; note that XVG2X is also symbolized simply as VG2. 63
y 10 y 8 y 7 y 6 a ₁ a 8 a ₂ a 5 a 4 a 6 a ₆ a 7 a 9 640.744 y 9 759.884 a ₉ 894.083 a ₄ y 6 y 7 y 10 [XVG₂ + Na] + a ₁ a ₂ a ₃ 231.255 328.374 385.435 484.550 506.570 a ₅ 605.696 y 8 662.746 a ₇ 737.848 a ₈ 794.9 859.029 966.025 100 200 300 400 500 600 700 800 900 1000 m/z Figure 4.6 MALDI-MS 2 spectrum of sodium cationized XVG2 peptide (m/z 966.025) acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum. X designates the propargyl-substituted end group of the peptide. The calculated monoisotopic mass of protonated XVG2 is 972.539114 Da. Figure 4.5 shows the MALDI-ToF/ToF mass spectrum of XVG2. The most abundant peak is due to the quasi-molecular ion at m/z 996.061, [XVG2+Na]+; the other ions are incomplete sequences. Figure 4.6 shows the MALDI-MS 2 spectrum of [XVG₂+Na]+. It corroborates the sequence XVPGVGVPGVGNH2, in which the N-terminal end group is acetylated propargyl glycine, C₇N₁H₈O₂ (138 Da), and the C-terminus is NH2. However, XVG2X and XVG2 have the same sequence up to the C-terminus; XVG2 has a truncated C- 64
terminus which lack the final propargyl glycine residue. The intensity of fragments y 9 and a 6 are again high because of the enhanced cleavages occurring N-terminal to proline. The major difference between the two MS 2 spectra is that a complete an series, from a1 until a9, is observed for the XVG2 peptide, instead of the complete yn series observed for XVG2X, pointing out that the sodium ion is now preferably attracted at the N-terminus. (B) [VG₂+2H]+² 534.7 (C) [VG₂+H]+ 1068.6 [VG₂+Na]+ 1090.6 [VG₂+H+Na]+² 545.7 [VG₂+2Na]+² 556.7 530 550 m/z [VG₂+K]+ 1126.6 (A) 1080 1100 m/z 200 400 600 800 1000 m/z Figure 4.7 A) ESI-MS spectrum of VG2 peptide dissolved in ammonium acetate buffer (ph=6.64) at 0.01 mg/ml plus 10 % (v %) of MeOH, acquired with the QIT mass spectrometer. B, C) Expanded views of the m/z regions containing B) doubly charged ions (orange box) and C) singly charged ions (purple box). 65
4.3.2 Characterization of VG2 by ESI-QIT-MS and ESI-Q/ToF-MS and MS n The best signal intensities in ESI-QIT mass spectra were achieved with VG₂ solutions in aqueous ammonium acetate buffer containing 10% (v %) MeOH (Figure 4.7). The most abundant peaks in Figure 4.7 are the doubly charged ions, which include the doubly protonated peptide at m/z 534.7, [VG₂+2H]+², and the quasi-molecular dications at m/z 545.7, [VG₂+H +Na]+², and m/z 556.7, [VG₂+2Na]+². The intensities of the singly charged ions, which are the protonated ion at m/z 1068.6, [M+H]+, and the quasi-molecular ions at m/z 1085.64, [M+NH4]+, and m/z 1090.648, [M+Na]+, are very low. The CAD spectrum of the doubly sodiated peptide [VG₂+2Na]+² at m/z 556.7, (Figure 4.8A) shows singly sodiated yn and an fragment series, which agree with those observed upon the MALDI-MS 2 fragmentation of [VG2+Na] + (Figure 4.4). Consecutive CAD-MS 3 on the [VG₂+Na]+ fragment at m/z 1090.6 from MS 2 of [VG₂+2Na]+ 2 (Figure 4.9) gives rise to the same yn and an series. The y9 and a6 fragments in the CAD-MS 2 spectrum of [VG2 +2Na] +2 were also selected for MS 3 experiments, which gave rise to the spectra shown in Figure 4.10 and Figure 4.11, respectively. The fragmentation patterns in these MS 3 spectra fully confirm the VG2 peptide sequence. The y9 fragment mainly yields truncated yn product ions (from y8 to y2), while the a6 fragment mainly yields truncated an product ions (from a5 to a3). In both cases, the predominant fragment series in the MS 3 spectra still contain one original end group, indicating that the end group structures provide a favorable coordination site to the Na + charge. 66
601.1 640.1 658.1 855.3 700.2 959.4 739.2 757.2 798.3 814.3 C₇ +17 C₈ +17 C₉ +17 954.4 1011.5 1113.5 C₁₀ +17 [VG₂+2Na]+ A [VG₂+ Na] + 1090.6 y 10 y 8 y 7 y 6 y 5 y 4 y 9 a ₂ a ₄ a ₆ a ₉ 556.8 a 4 a 6 291.1 348.1 445.2 484.1902 y y 9 4 y 2 y y 6 3 y 7 a 9 y 10 601.4 640.4 854.5 700.4 893.5 953.0 1072.6 B 200 300 400 500 600 700 800 900 1000 m/z z 10 y 8 y 7 y 6 c 6 c 7 a 7 c 5 a c ₂ 4 [VG₂+Na+17] +² 553.6 a ₆ c 8 c 9 c 10 [VG₂+Na+17]+ 1107.5 z ₁₀ [VG₂+ Na] + 1090.6 C₄ +17 545.2 C₅ +17 602.1 y 6 a ₆ y 7 C₆ +17 701.2 a ₇ y 8 500 600 700 800 900 1000 m/z Figure 4.8 CAD (A) and ETD (B) mass spectra of doubly sodiated VG2 ([VG2+2Na] +2 at m/z 556.7) acquired on the ESI-QIT mass spectrometer. The ETD ion reaction time was 400 ms. All fragments are singly sodiated unless marked by which denates sodiation plus one H/Na exchange (two Na+ ions overall). 67
y 10 [VG₂+ Na] + 1090.6 y 8 y 7 y 6 y 5 y 4 y 9 a ₂ a ₄ a ₆ a ₉ y 9 a 6 a y y y a 9 4 y 7 5 6 4 445.2 484.1902 640.4 854.5 544.2 601.4 700.4 893.5 y 10 953.6 1045.6 1072.6 200 300 400 500 600 700 800 900 1000 m/z Figure 4.9 CAD-MS 3 spectrum of the [VG2+Na] + fragment (m/z 1090) from [VG2+2Na] +2, acquired on the ESI-QIT mass spectrometer. The ETD spectrum of doubly sodiated peptide VG₂, i.e. [VG₂+2Na]+² at m/z 556.7, (Figure 4.8B) shows a contiguous cn +17 series (n = 4-10) as well as a few y n (n= 6-8) and a n (n= 6,7) fragments. All these ions carry one sodium ion. In addition, the doubly sodiated z 10 fragment is observed. The ion [VG2+Na+H] +2 (m/z 545.6) with mixed charges was also investigated by ETD (Figure 4.12). The resulting spectrum shows a complete c-type series from c 5 to c 10, including both closed-shell fragments as well as radical ions (missing one H ). All these fragments are observed in sodiated form and most of them also appear in protonated form. Similarly, the ETD spectrum of [VG2+2K] +2 (m/z 572.6) includes a complete c n series from n=5 to 10 (Figure 4.13). In addition, two zn 68
291.2 305.2 348.2 404.3 545.3 558.3 476.4 601.4 715.5 758.5 701.4 854.5 ions are observed. In contrast to the Na + containing precursor ions, the potassiated precursor ion only produces c-type fragments with two K + (except for c5). y ₃ y ₂ y 8 y 7 y 6 y 5 y 4 y 9 a y 4 445.3 a a a a 8+1 658.4 a CONH₂ 810.4 NH₃ 837.5 y 7+1 y 6 a₉ +1 y 8+1 y 2 a 4 y 3 a 5 y 5 a ₇ 200 300 400 500 600 700 800 m/z Figure 4.10 CAD-MS 3 spectrum of the y9 (m/z 854.5) fragment from [VG2+2Na] +2 (m/z 556.7), acquired on the ESI-QIT mass spectrometer. Representative structures of the fragments present in the ETD spectra are provided in Figure 4.14. Long ion-ion reactions times (400-600 ms) had to be used to obtain these spectra. At shorter reaction times, the fragmentation extent was very low. Long ion-ion reaction times in the QIT allow intra- and intermolecular ion-molecule reactions and radical-site reactions to occur in the precursor ions and their charge-reduced forms as well as in the initially formed fragments. H transfers can account for the 69
230.8 272.1 306.9 385.9 503.1 PGV+NH₃+H+ 623.3 484.2 VPGVGV-CO+Na+ 541.3 569.1 640.3 significant number of radical ions present in the ETD spectra. Ion-molecule reactions could explain the 17 Da adducts which may involve the addition of OH or NH₃, as shown in Figure 4.14 (the QIT does not have the resolution and mass accuracy needed to distinguish such isobaric adducts). The predominance of N-terminal fragments from all precursor ions further suggests more favorable energetics for charge retention at the N- terminus..1 a 1 404.1 a3 a 4 a₅ b₅ PGVGV-CO+Na+ a 6 404.1 a 4 b₅ a 3 +1 a 5 -NH₃ a 1 200 300 400 500 600 m/z Figure 4.11 CAD-MS 3 spectrum of the a6 (m/z 640.3) fragment from [VG2+2Na] +2 (m/z 556.7), acquired on the ESI-QIT mass spectrometer. 70
c 6 c 7 c 8 c 9 c 5 [VG₂+H+Na]+ 1091.5 y 5 544.1 562.0 c₅ 585.1 684.2 662.2 c₆ c₇ c₈ 782.3 798.2 817.3 839.3 916.4 c₉ 937.4 c₁₀ 995.5 [VG₂+2H]+ 1069.5 1107.5 600 700 800 900 1000 m/z [VG₂+2Na]+ 1113.5 500 600 700 800 900 1000 m/z Figure 4.12 ETD spectrum of [VG2+H+Na] +2 (m/z 545.6) acquired on the ESI-QIT mass spectrometer, using an ion-ion reaction time of 600 ms. All fragments are singly sodiated unless marked by H, which denotes ions with proton charges (no Na + ). It is noteworthy that N-C α cleavages at the proline residue are observed in the ETD spectra of Na+ containing VG2 (c6 +17 in Figure 4.8 B and c6/c6 in Figure 4.12), but not in the ETD spectrum of potassiated VG2 (Figure 4.13). Several studies have reported that fragmentation at the Pro residue does not take place with multiply protonated peptides because it requires the cleavage of two bonds in the radical intermediates formed after electron transfer. 57,133 The data in Figures 4.8 B and 4.12 clearly document that the use of Na + cationization (and possibly long reaction times) enables this reaction, increasing the sequence information that can be gained by ETD- MS 2. 71
z ₁₀ z ₈ c 6 c 7 c 5 c 8 c 9 c 10 [VG₂+2K] + 1145.5 c₉ 992.4 z₁₀ c₁₀ 991.4 1049.4 [VG₂+2H+NH₃] + 1086.5 572.5 c₅ c₅ 602 640.1 z₈ 795.2 c₇ c₈ 836.2 893.3 949.3 200 400 600 800 1000 m/z Figure 4.13 ETD spectrum of doubly potassiated VG2 ([VG2+2K] +2 at m/z 572.6), acquired on the ESI-QIT mass spectrometer using an ion-ion reaction times of 600 ms. An asterisk indicates singly charged ions with two K + (one K + charge and one H/K exchange). The sample analyzed by ESI-Q/ToF-MS was prepared by dissolving VG2 in ammonium acetate buffer (ph=6.64) at a concentration of 0.01 mg/ml and adding 10 % (v %) MeOH. Figure 4.15 shows the ESI-MS spectrum obtained with the Synapt Q/ToF mass spectrometer. The most abundant peaks in Figure 4.15 correspond to the same ions as those present in the ESI-QIT mass spectrum; the doubly charged ions include m/z 534.8 ([VG₂+2H]+² ) and the quasi-molecular ions at m/z 545.7 ([VG₂+H +Na]+² ) and m/z 556.7 ([VG₂+2Na]+²). The intensities of the singly charged ions, which include m/z 72
1068.6 ([M+H]+) and the quasi-molecular ions at m/z 1085.6 ([M+NH4]+ ) and m/z 1090.6 ([M+Na]+ ), are very low. The ESI-Q/ToF mass spectrum also contains several singly protonated fragment ions, like y1, y4, a5 and y9, which are ascribed to the CAD of [M+2H] +2 in the interface region between the ion source and the vacuum system. c₁₀ +OH c₁₀ +NH₃ c₆ z₁₀ Figure 4.14 Plausible structures of the ETD fragments from doubly charged VG2. Note that c6 is formed by cleavage of bonds within a proline residue. 73
490.3 Figure 4.16 shows that IM-MS removes chemical noise and separates the intact peptide with one and two charges from fragments. The drift time for the singly charged ions is about 4 ms and the drift time for the doubly charged ions is about 1 ms. In either charge state, each intact peptide ion shows a single drift time distribution (see drift time chromatogram for [VG2+Na] + in Figure 4.16 D), consistent with only one sequences and one structure. y 4 y 1 y 9 a 5 423.2480 [VG₂+2H]+² 534.8071 [VG₂+2Na]+² 556.7947 273.1313 526.3 646.3762 170.1024 568 832.4940 410.3 815.5 719.4369 899.5350 [VG₂+Na]+ 1090.5890 100 300 500 700 900 1100 m/z Figure 4.15 ESI-MS spectrum of VG2 dissolved in ammonium acetate buffer (ph=6.64) at 0.05 mg/ml + 1% (v %) of MeOH, acquired with the Synapt Q/ToF mass spectrometer. 74
(A) m/z 1000 $ 4.15 ms (C) $ @ [VG₂+Na] + 0.96 ms 1090.5768 500 [VG₂+H] + 1068.6185 [VG₂+NH 4 ] + 1085.6185 (B) 0.90 1.81 2.71 3.61 4.51 drift time (ms) @ [VG₂+2H]+² 534.7646 [VG₂+H+Na]+² 545.7499 [VG₂+2Na]+² 556.7427 [VG₂+3Na-H]+² AA_10162013_XVG2X_IM.raw AA_10162013_XVG2X_IM.raw : 1 : 1 (D) 1070 4.15 1090 m/z 567.7576 [VG₂+H+K]+² 553.7700 535 550 565 m/z 2.75 3.50 4.25 5.00 5.75 Figure 4.16 A) 2-D IM-MS plot (m/z vs. drift time) of VG2 dissolved in ammonium acetate buffer (ph=6.64) at 0.01 mg/ml with 10 % (v %) MeOH, acquired with the Synapt Q/ToF mass spectrometer. B) Mass spectrum extracted from IM region @ of the 2-D diagram. C) Mass spectrum extracted from IM region $ of the 2-D diagram. D) Drift time chromatogram (mobilogram) for [VG2+Na] + (m/z 1090.58). 4.3.3. Characterization of VG2 by Using Surfactant as a Solvent Surfactants, such as n-octyl-β-d-glucoside (β-c₈g₁) and n-dodecyl-α-dmaltoside (α-c₁₂g₂), have been used to dissolve lipid membranes and polypeptides. 134-137 In this study, these surfactants were used to dissolve the hydrophobic VG2 peptide in MeOH or H2O. Each surfactant was also separately dissolved in MeOH or H2O and directly injected in the ESI source of the Q/ToF instrument to determine the background ions formed without adding in on the peptide. Because α-c₁₂g₂ gave the least 75
background ions, it was chosen to dissolve the peptide in all ensuing IM-MS experiments. (B) [VG₂+2Na]+² 556.7072 [VG₂+H+K]+² (C) [VG₂+Na]+ 1090.4115 [VG₂+Na]+ 1090.4115 553.7000 [VG₂+NH₄]+ [VG₂+K+Na]+² 564.6932 [VG₂+H]+ 1068.4420 1085.4738 [VG₂+K]+ 1106.3684 1030 1050 1070 1090 1110 1130 1150 1170 m/z 530 540 550 560 570 580 m/z (A) [VG₂+2Na]+² 556.7072 [VG₂+H]+ 1068.4420 [ +H]+ 171.1490 327.0498 427.6749 719.3245 100 200 300 400 500 700 800 900 1000 m/z Figure 4.17 A) ESI-MS spectrum of VG2 dissolved at the concentration of 0.05 mg/ml in aqueous 0.01 mg/ml n-dodecyl-α-d-maltoside (α-c₁₂g₂) containing 10 % (v %) MeOH. The spectrum was acquired with the Synapt Q/ToF mass spectrometer B) Expanded view of the m/z region of doubly charged ions (green box). C) Expanded view of the m/z region of singly charged ions (purple box). When the concentration of the surfactant solution is the same as the concentration of the VG2 peptide, both VG2 and the surfactant components are detected. On the other hand, when the concentration of VG2 peptide is higher than that of the surfactant, only VG2 ions are detected in the ESI mass spectrum and the IM-MS plot. The n-dodecyl-α-d- 76
maltoside (α-c₁₂g₂) ions formed by ESI of directly injected surfactant are [C₁₂G₂+Na]+ at m/z 533.38 and [(C₁₂G₂)2+Na]+ at m/z 1043.8237. m/z 1000 500 1090.4115 y₁₀ y₉ a₇ y₅ a₅ a₁₀ y₈ y₇ y₄ y₆ 1.0 2.0 3.0 4.0 5.0 drift time (ms) Figure 4.18 2-D IM-MS 2 plot (m/z vs. drift time) of [VG2+Na] + (m/z 1090), acquired with the Synapt Q/ToF mass spectrometer from a VG2 solution (0.05 mg/ml) in surfactant containing 10 % (v %) MeOH. Figure 4.17A shows the ESI-MS spectrum of VG2 dissolved in surfactant solution at the concentration of 0.05 mg/ml + 10 % MeOH. The singly charged ions present include the protonated peptide at m/z 1068.4420 ([VG2+H]+) and quasi-molecular ions at m/z 1085.4738 ([VG2+NH4]+), 1090.4115 ([VG2+Na]+) and 1106.3684 ([VG2+K]+). Figure 4.17C attests that because the concentration of the peptide is higher than that of the surfactant (0.01 mg/ml), the [(C₁₂G₂)2+Na]+ ion at m/z 1043.8237 was not detected. The doubly charged peptide ions in Figure 4.17 B include m/z 553.7000 ([VG₂+2H]+²), 77
m/z 556.7072 ([VG₂+2Na]+²) and m/z 564.6932 ([XVG₂+Na +K]+²). The peak at m/z 533.38 corresponding to [C₁₂G₂+Na]+ was also not detected. y 10 y 8 y 7 y 6 y 5 y 4 [VG₂+ Na] + 1090.4115 y 9 a ₂ a ₄ a ₆ a ₉ a 6 640.2772 y 4 y 2 y 3 290.7 347.7 404.1778 445.1686 a 4 484.1902 y 5 y 6 y 7 544.2211 601.2219 700.2660 y 8 757 y 9 854.3344 a 9 893.3547 y 10 953.0 1072.3890 100 200 300 400 500 600 700 800 900 1000 m/z Figure 4.19 Mass spectrum extracted from the 2-D IM-MS 2 plot (m/z vs. drift time) of sodium cationized VG2 (m/z 1090), formed by ESI of a 0.05 mg/ml solution of the peptide in 0.01 mg/ml aqueous surfactant containing 10 % (v %) MeOH. Comparison of the ESI-Q/ToF mass spectra and IM-MS plots of VG2 obtained by using ammounium acetate buffer (Figure 4.16) and surfactant solution (Figures 4.17, 4.18) reveals that the intensity of singly charged ions is higher with the surfactant solution, as is the corresponding drift time (4.60 ms vs. 4.15 ms with the buffer solution). Hence, the surfactant solution improves the ESI efficiency of VG2 and reduces its compactness (folding), presumably by decreasing the extent of intramolecular hydrogen bonds. Figure 4.18 shows the 2-D IM-MS 2 plot (m/z vs. drift time) of [VG2+Na] + at m/z 78
1090 and Figure 4.19 shows the MS 2 spectrum extracted from this plot. The same yn and an fragment ions are detected if the buffer is used to dissolve the VG2 peptide. Using the surfactant solution of the hydrophobic peptide affected the intensity and drift time but did not affect the fragmentation pathways. All MS 2 spectra of VG2 (MALDI-MS 2, ESI-QIT- MS n, ESI-IM-MS 2 ) confirmed the VG2 peptide sequence and end groups. + ATRP Dimethyl 2,6- dibromoheptanedioate tert-butyl acrylate Poly (tert-butyl acrylate) dibromide NaN 3 CF 3 CO 2 H (TFA) Poly (acrylic acid) diazide Poly (tert-butyl acrylate) diazide Scheme 4.1 Synthesis of azide-functionalized PAA. 4.4 Characterization of PAA Polymer Polyacrylic acid is a nontoxic and biodegradable polymer. A summary of the poly acrylic acid (PAA) structures analyzed is given in Scheme 4.1. The synthesis of the PAA started with atom transfer radical polymerization (ATRP) of tert-butyl acrylate, using dimethyl 2, 6-dibromoheptanedioate as telechelic initiator to form poly(tert-butyl acrylate) dibromide. Subsequent reaction with sodium azide led to poly(tert-butyl 79
acrylate) diazide which was hydrolyzed by trifluoroacetic acid to form poly(acrylic acid) diazide (Scheme 4.1). The final product has diazide end groups [the molecular mass of the end groups, 2*N3, is 84.018444 Da] and a central group composed of two methyl acrylate and one methylene moieties [the molecular mass of the central group, C9H14O4, is 186.08921 Da]. The total mass of the end groups and central group is 270.107654 Da. PAA PAA₁₄ 1301.7 1373.7 PAA₁₆ 1445.7 1517.8 PAA₁₈ 1589.7 1661.8 PAA₂₀ 1733.8 1805.9 PAA₂₂ 1877.9 1949.9 PAA₂₄ 2021.8 2093.9 PAA₂₆ 2165.9 2237.8 [PAA₁₉+Na]+ 1661.8 1300 1500 1700 1900 2100 [PAA₂₁+Na]+ [PAA₂₀+Na]+ 1805.9 1733.8 2300 m/z 1660 1680 1700 1720 1740 1760 1780 1800 m/z Figure 4.20 MALDI-MS spectrum of polyacrylic acid (PAA) in positive ion mode. The sample was dissolved in THF/ MeOH (50:50, v/v %) at 20 mg/ml. SA matrix (20 mg/ml) and NaTFA cationizing salt (10 mg/ml) solutions were mixed (100:10, v/v %) and the sandwich method was used. 80
4.4.1 Characterization of PAA by MALDI-ToF/ToF-MS Figure 4.20 shows the MALDI mass spectrum of polyacrylic acid (PAA) obtained by using the MALDI-ToF/ToF instrument in positive ion mode. A major series with the polyacrylic acid repeat unit (C3H4O2, 72 Da) is clearly visible (labeled by PAAn). This series arises from sodiated oligomers of PAA. The m/z values of all these ions agree well with the composition (C3H4O2)n + C9H14O4N6 (270 Da), confirming that complete hydrolysis of poly(tert-butyl acrylate) (PtBA) to PAA took place and that the resulting PAA contained the desired central substituent and diazide end groups. Several minor distributions are observed due to exchange of the COOH proton with Na+ or K+. Ion intensities are low in Figure 4.20. PAA₁₈ PAA PAA₁₀ 989.145 1061.220 PAA₁₂ 1133.284 1205.380 PAA₁₄ 1277.533 1349.852 PAA₁₆ 1421.728 1493.738 PAA₂₀ 1565.838 1637.933 1710.004 1782.0 PAA₂₂ 1854.205 1927.294 PAA₂₄ 1999.374 2071.456 PAA₂₆ 2142.479 2215.733 PAA₂₈ 2287.4 2359.76 PAA₃₀ 2431.966 1000 1200 1400 1600 1800 2000 2200 2400 m/z [PAA₁₇-H] 1493.738 [PAA₁₈-H] [PAA₁₈-H] 1565.838 1637.933 1490 1510 1530 1550 1470 1590 1610 1630 1650 m/z Figure 4.21 MALDI-MS spectrum of polyacrylic acid (PAA) in negative ion mode. The sample preparation was the same as in positive mode. 81
Figure 4.21 shows the MALDI mass spectrum of polyacrylic acid (PAA) obtained with the MALDI-ToF/ToF instrument in negative ion mode. A major series with the polyacrylic acid repeat unit (C3H4O2, 72 Da) is clearly visible, especially in the expanded view of Figure 4.21. This series arises from deprotonated oligomers of PAA whose m/z values agree well with the composition (C3H4O2)n + C9H14O4N6 (270 Da). Ion intensities of PAA are much higher in negative ion mode than in positive ion mode pointing out that proton loss from PAA during MALDI is more efficient than the gain of a sodium cation. Both positive and negative ion mode MALDI-MS of PAA confirm the formation of a polymer with diazide end groups and a C9H14O2 central group. MS 2 experiments could not be performed, however, because of the low intensities in positive mode and the low fragmentation efficiency in negative mode. 4.4.2 Characterization of PAA by ESI-Q/ToF-MS ESI-MS of PAA leads to very complex spectra showing ions in multiple charge states in both positive and negative modes. ESI of PAA mainly produces doubly charged ions in positive mode and doubly or triply charged ions in negative ion mode. With ion mobility mass spectrometry, PAA polymer ions can be separated according to their charge and also from chemical and contamination noise with repeat units of 28 and 44 Da. In positive ion mode, a major series of doubly charged ions with the polyacrylic acid repeat unit (C3H4O2, 72 Da/2=36 m/z units distance between successive oligomers) is clearly visible in the IM-MS spectrum extracted from the mobility region of +2 ions (Figure 4.22). This series arises from the addition of two potassium ions to PAA 82
oligomers. The m/z values of all these ions agree well with the composition (C3H4O2)n + C9H14O4N6 (270 Da). Other, minor distributions are also observed due to exchange of the COOH protons with Na or K. m/z [PAA₁₃+2K]+² 642.1671 [PAA₁₄+2K]+² 678.1825 [PAA₁₅+2K]+² 714.1971 1500 649.1772 [PAA₁₃+ K+H]+² [PAA₁₃+K+ 3Na-2H]+² 659.1970 661.1439 [PAA₁₃+ 3K-H]+² [PAA₁₄+K+ 3Na-2H]+² 685.2030 [PAA₁₄+ K+H]+² 695.2244 697.1753 [PAA₁₄+ 3K-H]+² 1000 500 [PAA]+² 0.9 1.81 2.71 3.61 4.51 5.42 6.32 drift time (ms) 7.22 642 652 662 672 682 692 702 712 m/z Figure 4.22 2-D IM-MS plot (m/z vs. drift time) of PAA dissolved in THF/MeOH (50:50, v/v %) at 0.1 mg/ml plus 1% (v %) formic acid, acquired with the Synapt Q/ToF mass spectrometer in positive ion mode. B) Mass spectrum extracted from the circled mobility region, where [PAA+2K] +2 ions dominate. In negative ion mode, -3 and -2 ions are mainly observed. The major series in the mass spectrum extracted from the IM-MS mobility region of triply charged ions (Figure 4.23) arises from PAA oligomers that lost three protons and exchanged one additional proton with a K ion. On the other hand, the dominant ions in the mass spectrum extracted from the IM-MS mobility region of doubly charged ions (Figure 4.23) arise from doubly deprotonated PAA oligomers of PAA. As expecteded, triply charged ions have lower drift times than the doubly charged ions. The m/z values of all deprotonated ions agree 83
501.1274 545.1165 well with the composition (C3H4O2)n + C9H14O4N6 (270 Da). Several additional distributions are observed in the -3 and -2 charge states due to exchanges of COOH protons with Na or K. Both MALDI MS and ESI MS confirm the expected PAA composition and end groups. m/z 1000 [PAA] ³ [PAA] ² 500 [PAA₁₈-4H+K] ³ 533.7695 538.4338 [PAA₁₈-5H +Na+K] ³ 541.0900 [PAA₁₈-5H +2K] ³ 546.4132 [PAA₁₉-4H +Na] ³ 553.7330 3.61 5.42 2.71 7.22 9.03 [PAA₁₉-4H+K] ³ 557.7715 drift time (ms) [PAA₁₉-5H +Na+K] ³ [PAA₁₉-5H 565.0969 +2K] ³ 570.4226 572.4179 AA_05282014_PAA_OLD_NE_1.raw : 1 576.9614 577.732 10.83 12.64 [PAA₂₀-4H+K] ³ 581.7605 [PAA₂₀ -4H +Na] ³ 534 542 550 558 566 574 582 m/z [PAA₁₁-2H] ² [PAA₁₀-2H] ² 530.1152 494.1078 [PAA₉- 7H+3K+2 Na] ² [PAA₁₀- 3H+Na] ² 505.1017 [PAA₁₀- 3H+K] ² 513.0893 [PAA₉- 7H+K+4 Na] ² 521.1089 [PAA₁₀- 7H+3K+2 Na] ² 537.1295 [PAA₁₁-3H+Na] ² 541.1156 [PAA₁₁ -3H+K] ² 549.0889 [PAA₁₀- 4H+2K] ² 497.1113 [PAA₁₀- 4H+Na+K] ² 560.0920 [PAA₁₂-2H] ² 566.1198 494 504 514 524 534 544 554 564 m/z Figure 4.23 2-D IM-MS plot (m/z vs. drift time) of diazide-terminated polyacrylic acid in negative ion mode. The sample was dissolved in THF/MeOH (50:50, v/v %) at 0.1 mg/ml plus 1% (v %) formic acid. B) Mass spectrum extracted from the circled region of -3 ions, where [PAA-3H+K] -3 anions dominate. C) Mass spectrum extracted from the circled region of -2 ions, where [PAA-2H] -2 anions dominate. 84
4.5 Characterization of PtBA-VG2 The synthetic route to the hybrid materials examined is outlined in Scheme 4.2. The synthesis of PtBA-VG2 and PAA-VG2 started with a click chemistry reaction involving azide-alkyne Huisgen cycloaddition between the azide groups of PtBA polymer and alkyne groups of VG2 peptide. In the presence of copper (I) acetate catalyst, 1, 3- dipolar cycloaddition takes place between the terminal alkynes and azides to form PtBA- VG2 hybrid material whose t-butyl esters were subsequently hydrolyzed with trifluoroacetic acid (TFA) to form PAA-VG2 hybrid material (Scheme 4.2). The click chemistry reaction can lead to a linear hybrid material with one azide and one alkyne end group or the chain ends may also react to form a cyclic hybrid material; the linear and cyclic structures have the same molecular mass and both could contain one or more constituent blocks. For simplicity, only the linear structures of the hybrid materials were drawn in Scheme 4.2. The PtBA-VG2 hybrid material in Scheme 4.2 possesses a triazole containing linker at the N-terminal side of VG2 peptide [the molecular mass of this group, C7O2N4H8, is 180.064696 Da], a VG2 peptide segment without the end groups [the molecular mass of VG2 peptide, C₃₈O₁₀ N₁₀ H₆₂, is 818.46489 Da], another triazole containing linker as the C-terminal side of VG2 peptide [the molecular mass of this linker, C5N5O1H7, is 153.06506 Da] and a center group containing the telechelic initiator used in the acrylate polymerization [the molecular mass of the center group, C9H14O4, is 186.08921 Da]. The mass of all substituents of the polyacrylate chain in one PtBA-VG2 block is equal to 1337.683841 Da. 85
+ PtBA Cu(I) acetate DMF,80 C VG₂ [PtBA VG₂] m CF 3 CO 2 H (TFA) [PAA VG₂] m Scheme 4.2 Synthesis of azide-functionalized PtBA-VG2 and PAA-VG2. 4.5.1 Characterization of PtBA-VG2 by MALDI-ToF/ToF-MS Figure 4.24 shows the MALDI mass spectrum of PtBA-VG2 in positive ion mode, using DCTB as matrix and KTFA as cationizing salt. The major series with the PtBA repeat unit (C7H12O2, 128.0777 Da), labeled by An, arises from potassiated ions of PtBA- VG2 oligomers and the m/z values of all these ions agree well with the composition (C7H12O2)n + C59H91O17N19 (1337.68 Da). A second series with the PtBA repeat unit is also clearly visible and this series arises from protonated ions of PtBA-VG2 oligomers 86
that underwent exchange of the N3 end group with a OCH3 group. This exchange could occur during the synthesis or during the sample preparation process. The intensity of PtBA-VG2 ions in positive ion mode is low indicating that the components of this hybrid material are difficult to ionize under MALDI conditions. A₁₁ A₁₂ A B A₆ 2145.2 A₇ 2273.3 A₈ A₉ 2529.4 A₁₀ 2785.6 2913.7 A₁₃ 2657.5 3041.8 A₁₄ 3169.8 A₁₅ 3297.9 A₁₆ 2401.3 3426.0 B B B B A₁₇ 3554.1 B B B B B B B A₁₈ A₁₉ 3682.1 3810.2 B B A₂₀ A₂₁ [A₁₀+K]+ 2657.5 [A₁₁+K]+ 2785.6 2000 2250 2500 2750 3000 3250 3500 3750 4000 m/z [A₁₂+K]+ 2913.7 [B₁₀+H]+ 2736.8 [B₁₀+H]+ 2864.9 2650 2700 2750 2800 2850 2900 m/z Figure 4.24 MALDI-MS spectrum of poly(tert-butyl acrylate-vg2 peptide) (PtBA-VG2) hybrid material acquired on the MALDI-ToF/ToF mass spectrometer in positive mode. The sample was dissolved in NH4OAc/ MeOH (50:50, v/v %) at 20 mg/ml. DCTB (20 mg/ml) and KTFA (10 mg/ml) were mixed in the ratio 100:10 (v/v %) and the sandwich method was used. 87
4.5.2 Characterization of PtBA-VG2 by ESI-Q/ToF-MS The ESI mass spectrum of PtBA-VG2 in positive ion mode is very complex, containing ions with multiple charge states. The PtBA-VG2 hybrid material is observed doubly and triply charged. With IM-MS, these charge states are easily separated (Figure 4.25). The mass spectrum extracted from the mobility separated doubly charged ions shows a dominant series with the PtBA repeat unit (C7H12O2, 128 Da), arising from doubly protonated PtBA-VG2 oligomers. Because these oligomers carry +2 charges, they appear every 128/2= 64 m/z units. The m/z values of all these ions agree well with the composition (C7H12O2)n + C59H91O17N19 (1337.68 Da). Ion series with the PtBA repeat unit are also present in the mass spectrum extracted from the mobility-separated triply charged region. Here, the charges are provided by 3 protons and successive oligomers differing by one PtBA repeat unit are 128/3 =42.67 m/z units apart from each other. The +3 region includes oligomers with the polyacrylic acid repeat unit (C3H4O2, 72 Da), which appear every 72/3=24 m/z units. There are attributed to partial COOtB COOH hydrolysis in the ammonium acetate solution used to dissolve the PtBA-VG2 sample. Essentially all PtBA-VG2 oligomers observed in +3 charge state underwent partial tertbutyl ester hydrolysis; oligomers with COOH pendants appear to protonate more easily than oligomers with completely esterified acrylate side chains, presumably because of more facile H-bonding. The structure of PtBA-VG2 will be discussed later in section 4.7. 88
m/z 4000 3000 [PtBA₆-VG₂+2H]+² 1054.26 [PtBA₇-VG₂+2H]+² 1118.2 [PtBA₈-VG₂+2H]+² 1182.2 2000 [PtBA-VG₂]+³ [PtBA-VG₂]+² 64 m/z 64 m/z 1000 2 4 6 8 10 12 14 16 drift time (ms) 1055 1075 1095 1115 1135 1155 1175 m/z [B₅A₃-VG₂ +3H]+³ 732.43 [B₄A₅-VG₂ +3H]+³ 737.76 42.7 m/z [B₆A₂-VG₂ +3H]+³ 751.12 [B₅A₄-VG₂+3H]+³ 756.45 [B₄A₆-VG₂ +3H]+³ 761.77 [B₇A₁-VG₂ +3H]+³ 769.78 [B₆A₃-VG₂+3H]+³ 775.13 [B₅A₅-VG₂ +3H]+³ 780.46 24.0 m/z [B₇A₂-VG₂ +3H]+³ 793.81 [B₆A₄-VG₂ +3H]+³ 799.11 730 740 750 760 770 780 790 800 m/z Figure 4.25 2-D IM-MS plot (m/z vs. drift time) of the PtBA-VG2 hybrid material. The sample was dissolved in NH4OAc/ MeOH (50:50, v/v %) at 0.1 mg/ml plus 1% (v %) formic acid. The mass spectra extracted from the +2 and +3 mobility regions are depicted beside and below the plot, respectively. 4.6 Characterization of PAA-VG2 According to Scheme 4.2, the PAA-VG₂ hybrid material has the same end groups and center substituent as the PtBA-VG2 hybrid material which contribute 1337.683841 Da to its mass. The difference between the two hybrid materials is in the polymer repeat 89
unit. If the structure of hybrid material is linear, the center and end groups consist of the azide end group (42.009222 Da), VG2 peptide (818.46489 Da) with its N-terminal (C₇O₂N4H₈, 180.064696 Da) and C-terminal (C5N2O1H7, 111.055823 Da) substituents and the polyacrylate initiator center group (C9H14O4, 186.08921 Da).!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 5100 5400 m/z! [A₁₅-VG₂+Na]+ 2441.3! [A₁₆-VG₂+Na]+ 2513.3! [A₁₇-VG₂+Na]+ 2585.3 [A₁B₈-VG₂ +Na]+ [A₁₀B₃-VG₂ +Na]+ [A₁₂B₂-VG₂ +Na]+ [A₁₄B₁-VG₂ +Na]+ [A₉B₄-VG₂ +Na]+ [A₂B₈-VG₂ +Na]+ [A₁₁B₃-VG₂ +Na]+ [A₁₃B₂-VG₂ +Na]+ [A₁₅B₁-VG₂ +Na]+ 2450 2470 2490 2510 2530 2550 2570 m/z Figure 4.26 MALDI-MS spectrum of polyacrylic acid-vg2 peptide (PAA-VG2) hybrid material acquired on the MALDI-ToF/ToF mass spectrometer in positive ion mode. The sample was dissolved in DMSO at 20 mg/ml; CHCA served as matrix and the sandwich method was used. An designates the number of PAA units and Bn the number of PtBA units. 90
4.6.1 Characterization of PAA-VG2 by MALDI-ToF/ToF-MS Figure 4.26 shows the MALDI-MS spectrum of PAA-VG2 acquired on the MALDI-ToF/ToF mass spectrometer in positive ion mode. The major series with the polyacrylic acid repeat unit (C3H4O2, 72 Da), labeled by! arises from sodiated oligomers of PAA-VG2. The m/z values of all these ions agree well with the composition (C3H4O2)n + C59H91O17N19 (1337.68 Da), confirming that a major fraction of PtBA underwent complete hydrolysis to PAA. Several minor distributions are observed due incomplete hydrolysis of the PtBA ester groups. Figure 4.27 shows MALDI-MS spectrum of PAA-VG2 acquired on the MALDI- ToF/ToF mass spectrometer in negative ion mode. The major series with the polyacrylic acid repeat unit (C3H4O2, 72 Da), labeled by *, arises from deprotonated oligomers of PAA-VG2. Their m/z values agree well with the composition (C3H4O2)n + C59H91O17N19 (1337.68 Da), reaffirming that PtBA underwent largely complete hydrolysis to PAA. Several minor distributions are also observed due to incomplete hydrolysis of PtBA to PAA, like in positive ion mode. In both modes, all ion series contain the correct center and end groups (1337.6 Da) and minor ion series from incomplete hydrolysis of PtBA to PAA are detected. 91
* * * * * * * * ** * * * * * * * * ** * * * * * * * ** * * * 1100 1400 1700 2000 2300 2600 2900 3200 3500 3800 4100 4400 m/z * * * [A₁₀-VG₂-H] 2057.0 [A₁₁-VG₂-H] [A₁₂-VG₂-H] 2129.3 2201.4 [A₅B₃-VG₂ -H] [A₇B₂-VG₂ -H] [A₉B₁-VG₂ -H] [A₆B₃-VG₂ -H] [A₈B₂-VG₂ -H] [A₁₀B₁-VG₂ -H] 2060 2080 2100 2120 2140 2160 2180 m/z Figure 4.27 MALDI-MS spectrum of the PAA-VG2 hybrid material acquired on the MALDI-ToF/ToF mass spectrometer in negative ion mode. The sample was dissolved in DMSO at 20 mg/ml; CHCA served as matrix and the sandwich method was used. An designates the number of PAA units and Bn the number of PtBA units. 4.6.2 Characterization of PAA-VG2 by ESI-Q/ToF-MS The ESI-MS spectra of PAA-VG2 are very complex, containing ions in multiple charge states, both in positive and negative ion modes. Under ESI conditions, PAA-VG2 forms doubly and triply charged molecular cations or anions. Using the ion mobility dimension, the hybrid material can be separated according to its charge as well as from 92
unconjugated polymer (PAA and PAA-PtBA) and incompletely hydrolyzed hybrid (PAA-PtBA-VG2) in various charge states. [PAA₁₄-VG₂+3H]+³ 783.043 [PAA₁₅-VG₂+3H]+³ 807.074 m/z [PAA₁₆-VG₂+3H]+³ 4000 831.076 3000 2000 [PAA-VG₂]+³ [PAA-VG₂]+² 780 790 800 810 820 830 m/z [PAA₁₀-VG₂+2H]+² 1030.06 [PAA₁₁-VG₂+2H]+² 1066.07 1000 [PAA]+ⁿ [PAA-PtBA]+ⁿ [PAA-PtBA-VG₂]+ⁿ 1.28 2.57 3.85 5.13 6.41 7.70 8.98 10.26 drift time (ms) [PAA₁₂-VG₂+2H]+² 1101.99 1030 1040 1050 1060 1070 1080 1090 1100 m/z Figure 4.28 2-D IM-MS plot (m/z vs. drift time) of PAA-VG2 hybrid material in positive ion mode. The sample was dissolved in NH4OAc at 0.01 mg/ml plus 10 % (v %) MeOH. The mass spectra extracted from the regions of doubly and triply charged PAA-VG2 are shown next to the plot. In positive ion mode (Figure 4.28), the IM-MS spectrum extracted from the region of doubly charged PAA-VG2 shows just one series with the PAA repeat unit (C3H4O2, 72 Da), which arises from doubly protonated oligomers with one constituent block [(PAA-VG2]1). Oligomers differing by one repeat unit are separated by 72/2= 36 m/z units, as expected for +2 ions; further, no PtBA content is detected beyond noise level. Very similar characteristics are present in the IM-MS spectrum extracted from the 93
mobility region of triply charged PAA-VG2. Again, one series is observed, arising from triply protonated oligomers of PAA-VG2, which appear every 72/3=24 m/z units. Their m/z values of correspond to hybrid material with one constituent block and no PtBA content. The m/z values doubly and triply protonated PAA-VG2 agree well with the composition (C3H4O2)n + C59H91O17N19 (1337.68 Da). A third mobility area in the 2-D IM-MS plot in Figure 4.28 contains PAA, PAA-PtBA and PAA-PtBA-VG2 with varying charges and for this reason it looks very complex and its ions have very low intensity. [PAA₁₅-VG₂-3H] ³ 804.8016 [PAA₁₆-VG₂-3H] ³ [PAA₁₇-VG₂-3H] ³ 828.7516 852.7764 m/z 3000 [PAA₁₅-VG₂- 4H+K] ³ 817.4041 [PAA₁₅-VG₂- 5H+2Na] ³ 819.3390 [PAA₁₂-VG₂-2H] ² 1099.6672 [PAA₁₅-VG₂- 6H+2K+Na] ³ 837.3564 [PAA₁₆-VG₂- 4H+K] ³ 841.4034 805 810 815 820 825 830 835 840 845 850 m/z [PAA₁₃-VG₂-2H] ² 1135.5880 2000 1000 [PAA-VG₂] ² [PAA-VG₂] ³ [PAA] ⁿ [PAA-PtBA] ⁿ [PAA-PtBA-VG₂] ⁿ 2.57 5.13 7.70 10.26 12.83 drift time (ms) [PAA₁₄-VG₂-2H] ² 11710.6580 [PAA₁₂-VG₂-3H+K] ² [PAA₁₃-VG₂-3H+K] ² 1118.5941 1154.5703 1100 1110 1120 1130 1140 1150 1160 1170 m/z Figure 4.29 2-D IM-MS plot (m/z vs. drift time) of PAA-VG2 hybrid material in negative ion mode. The sample was dissolved in NH4OAc at 0.01 mg/ml plus 10 % (v %) MeOH was added. The mass spectra extracted from the regions of doubly and triply charged PAA-VG2 are shown next to the plot. 94
The measurements in negative ion mode gave similar results. The major series in the IM-MS spectrum extracted from the region of doubly charged PAA-VG2 (Figure 4.29), whose members appear at intervals of 72/2= 36 m/z units, arises from doubly deprotonated oligomers with one constituent block. The same oligomers, triply deprotonated (every 72/3 = 24 m/z units), dominate in the spectrum extracted from the region of triply charged PAA-VG2. In both spectra, minor series due to one or more COOH COOX (X= K or Na) exchanges are also present. The m/z values of the major and minor ion series of doubly and triply deprotonated hybrid material corroborate the composition (C3H4O2)n + C59H91O17N19 (1337.68 Da). The third circled area in the IM- MS plot in Figure 4.29 includes PAA, PAA-PtBA and PAA-PtBA-VG2 with varying charges; for this reason, it looks very complex and noisy. In summary, both MALDI MS and ESI MS confirm the expected of PAA-VG2 composition and substituents incorporated in this material. 4.7 Structural Information from Collision Cross-Sections 4.7.1 Derivation of Collision Cross-Sections from Traveling Wave IM-MS Experiments From the drift time of an ion through the IM region, the corresponding collision cross-section can be derived which is an important physical property, characteristic of the ion s size and conformation (shape). With traveling wave IM-MS, there is no direct relationship between the measured drift time, t D, and the collision cross-section, Ω, as explained in chapter 2.6. Collision cross-sections are derived by calibrating the drift time scale with standards of known Ω, following a procedure that has been described in detail 95
Normalised CCS (Ω ) (Ų) in the literature. 65, 66 Generally, the calibration standards (calibrants) are ions whose Ω values were determined by the drift time ion mobility spectrometry (DTIMS) version of IM-MS, in which a constant electric field is applied to an IM chamber filled with He. The buffer gas in traveling wave IM-MS experiments is N2. However, it has been shown for ions of small and medium size (Ω < 600 Å 2 ) that collision cross-sections deduced from such experiments by using calibrant Ω values obtained with He the match within ±3% collision cross-sections measured directly with DTIMS. 65,130,138 The calibrants employed in this study were singly and doubly charged polyalanine oligomers. 139 The corrected collision cross-sections of these ions were plotted against the corrected drift times (arrival times) of the ions measured at the same traveling wave velocity, traveling wave height, and ion mobility gas flow setting used for the hybrid materials (Figure 4.30 and Table 4.1). 2100 Polyalanine WV=350, HW=8 1800 1500 1200 900 600 300 0 y = 373.33x 0.5507 R² = 0.9947 0 2 4 6 8 10 12 14 16 18 20 Corrected drift time (t D ) (ms) Figure 4.30 Plot of corrected drift times (arrival times) against corrected published crosssections for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. 96
Table 4.1 Drift time and collision cross-section data of singly and doubly protonated polyalanine oligomers, H-(Ala)n -OH. z a n mn-mer (Da) m/z td b (ms) td c Ω (Ų) reduced mass d Ω e (Ų) 1 2 160.08 161.09 1.25 1.2321 81 4.88250 395.482 1 3 231.15 232.16 1.54 1.5185 101 4.99827 504.825 1 4 302.11 303.12 2.19 2.1655 115 5.06295 582.239 1 6 444.15 445.16 3.33 3.3003 146 5.13307 749.428 1 10 728.24 729.25 6.49 6.4519 199 5.19351 1033.510 1 12 870.29 871.30 8.18 8.1384 223 5.20928 1161.670 1 16 1154.70 1155.70 11.54 11.4921 276 5.2294 1443.310 1 20 1438.91 1439.92 15.90 15.8465 337 5.24168 1766.440 1 22 1580.98 1581.99 18.08 18.0239 353 5.24618 1851.900 2 17 1225.42 613.72 3.07 3.0351 265 5.23298 693.370 2 18 1296.43 649.22 3.34 3.3041 276 5.23618 722.593 2 19 1367.45 684.74 3.58 3.5431 287 5.23906 751.806 2 20 1438.76 720.39 3.86 3.8222 297 5.24167 778.388 2 21 1509.51 755.77 4.11 4.0712 308 5.24402 807.579 2 22 1580.54 791.28 4.42 4.3803 317 5.24617 831.517 2 23 1651.55 826.78 4.75 4.7045 327 5.24813 858.069 2 24 1722.59 862.30 4.98 4.9336 337 5.24994 884.614 2 25 1793.61 897.82 5.32 5.2757 348 5.25160 913.778 2 26 1864.64 933.33 5.71 5.6619 358 5.25314 940.312 a For the doubly charged ions, CCS values were taken from reference 139. For the singly charged ions, the CCS values were calculated in this study using the Materials Studio and MOBCAL (projection approximation) programs. The CCS of singly charged ions from reference 139 did not fit into the same line as the CCSs of the doubly charged ions (see Appendix). b Measured drift time c t D` = t D - [ 1.41 m z 1000 ] d Reduced mass= m ion m N m ion +m N, where m ion and m N are the masses of the polyalanine ion (see Table) and the drift gas molecule (N2, 28 Da), respectively. e Obtained via Ω`= Ω*( m ion m N m ion +m N /z) 97
Table 4.2 Comparison of published collision cross-sections for different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of Figure 4.30. z m ion (Da) m/z t D (ms) t D a (ms) Reduced mass b Ω c (Ų) Ω d (Ų) Published Ω (Ų) % error e 9 8569.15 952.128 5.00 4.956 5.2838 901.416 1535 1649-7 10 8570.13 857.013 4.36 4.319 5.2838 835.570 1581 1732-10 11 8571.27 779.206 3.98 3.941 5.2838 794.459 1654 1802-9 13 12367.50 951.345 5.00 4.957 5.2865 901.418 2217 2391-8 14 12368.50 883.466 4.62 4.578 5.2865 862.843 2285 2473-8 15 12369.50 824.635 4.23 4.190 5.2865 821.709 2332 2579-11 16 12370.40 773.150 4.10 4.061 5.2865 807.709 2445 2679-10 17 12372.30 727.784 3.85 3.812 5.2865 780.066 2509 2723-9 18 12374.10 687.448 3.59 3.553 5.2865 750.426 2555 2766-8 19 12375.60 651.348 3.33 3.294 5.2865 719.788 2587 2800-8 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure 4.30 (y=373.3 x 0.5507 ) d Exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 The validity of the calibration curve in Figure 4.30 (y=373.33 x 0.5507 ; where y is the normalized CCS and x is the corrected drift time) was tested by using it to determine the CCS of several charge states of the proteins ubiquitin and cytochrome C (equine), which were previously measured by DTIMS. 140,141,142 Table 4.2 shows the drift times measured in this study and experimental CCSs derived from these values by using the calibration plot of Figure 4.30, along with the published CCSs of the ubiquitin and cytochrome C. The difference between published CCS and experimental CCS ranges between -7 and -11%. Using CCS reference data obtained with He to calibrate the drift 98
time scale of experiments performed in N2 appears to slightly underestimate large collision cross-sections (> 1600 Ų). Given the relatively small size of the PtBA-VG2 and PAA-VG2 oligomers analyzed in this study, the error in their CCS values obtained from the calibration plot of Figure 4.30 should be < 7%. 1054.6802 6.41 1054.1785 1055.1818 1055.6718 1056.1859 [(PtBA₆-VG₂)₁+2H]+² m/z 1054 1054 1056 m/z 1182.7728 1183.2789 2.00 4.00 6.00 8.00 10.00 drift time (ms) 7.70 1182.2794 1183.7726 1184.2535 1184.7472 [(PtBA₈-VG₂)₁+2H]+² m/z 1182 1182 1184 m/z 2.00 4.00 6.00 8.00 10.00 drift time (ms) Figure 4.31 IM-MS chromatogram (drift time distribution) of the ions at m/z 1054 and m/z 1182 from PtBA-VG2. The insets show the mass spectra extracted from the main peaks. 99
Doubly and triply protonated PtBA-VG2 oligomers were isolated for IM-MS analysis by the Synapt instrument under the following conditions: trap collision energy at 6 ev, transfer collision energy at 4 ev and low mass resolution at 11. Figure 4. 31 shows the IM-MS chromatograms of mass-selected [(PtBA6-VG2)1] +2 at m/z 1054 and [(PtBA8- VG2)1] +2 at m/z 1182. Both include a single drift time distribution of +2 ions with one PtBA-VG2 block, consistent with one structure for such oligomers. There is a broad shoulder with low intensity at lower drift times, which is attributed to higher charge states containing more than one PtBA-VG2 constituent blocks. Similar results are obtained for the +3 ions of PtBA-VG2. Table 4.3 summarizes the drift times of nine doubly or triply protonated PtBA- VG2 oligomers. In all cases, one single +2 or +3 ion drift time is observed, in agreement with one architecture for PtBA-VG2 hybrid material with one constituent PtBA-VG2 block. The drift times for the triply charged ions are lower than those for the doubly charged ions because higher charge states move faster inside the triwave IM area. Table 4.3 also lists the collision cross-sections derived for the nine doubly and triply protonated PtBA-VG2 oligomers by using polyalanine as a CCS calibrant. The experimental CCSs for the doubly charged ions range from 365 Š2 to 438 Ų for oligomers having between 4 to 10 PtBA repeat units. For triply charged ions, the CCS range is from 398 Ų to 458 Ų for oligomers containing between 5 to 9 PtBA repeat units. As expected, the CCS of the same oligomer increases with increasing charge state due to stronger charge repulsion forces in the structure carrying more charges. 100
Table 4.3. Experimental collision cross-sections of nine doubly and triply protonated PtBAn-VG2 oligomers, derived using the calibration plot of Figure 4.30. z n m n-mer (Da) m/z t D (ms) t D` (ms) Reduced mass Ω' (Ų) Ω (Ų) 2 4 1850.09 926.053 5.50 5.5290 5.2528 957.347 365 2 6 2106.35 1054.185 6.41 6.4411 5.2576 1041.323 396 2 7 2234.48 1118.246 7.05 7.0820 5.2596 1097.172 417 2 8 2362.54 1182.280 7.70 7.7330 5.2614 1152.070 438 2 10 2618.70 1310.359 8.72 8.7548 5.2644 1233.000 468 3 5 a 2194.30 732.443 3.08 3.1057 5.2590 696.822 398 3 6 a 2322.48 775.168 3.33 3.3564 5.2608 727.267 415 3 7 a 2450.51 817.844 3.59 3.6172 5.2625 757.858 432 3 9 a 2706.67 903.228 4.00 4.0286 5.2653 804.181 458 a the triply protonated oligomers also contain 3 hydrolyzed PAA repeat units. Several oligomers of PAA-VG2 were also examined by IM-MS. Figure 4. 32 shows the IM-MS chromatograms of mass-selected m/z 1030 and 1102 which correspond nominally to doubly protonated [(PAA10-VG2)1] and [(PAA12-VG2)1], respectively. Three peaks with three distinct drift times are observed for both ions, the middle components having the highest intensity. The isotope patterns and m/z values in the mass spectra extracted from the mobility-separated peaks reveal that the center components drifting at 5.42 ms (top) and 5.96 ms (bottom) arise from [(PAA10-VG2)1 +2H] +2 and [(PAA12- VG2)1 +2H] +2, respectively. Their narrow peak shapes are consistent with one architecture which could be linear or cyclic (vide infra). Conversely, the components drifting at 3.88 ms (top) and 4.06 ms (bottom) are identified as quadruply charged ions of oligomers with two constituent PAA-VG2 blocks., viz. [(PAA10-VG2)2 +4H] +4 and 101
1104.0826 1104.5350 1103.5817 1102.0669 1103.0807 1030.0367 1031.5486 1032.0330 1032.5176 1031.0522 [(PAA12-VG2)2 +4H] +4, respectively. Finally, the most slowly drifting components, appearing at 6.95 ms (top) and 7.13 ms (bottom) are assigned to mixtures of [PAA6 +K] + plus [PAA24 +Na+K] +2 (top) and [PAA11 +K] + plus [PAA26 +Na+K] +2 (bottom). Comparing the drift times of the three isobaric components of m/z 1030 and 1102 reveals that [(PAA-VG2)2 +4H] +4 ions with two blocks have the highest drift velocities and lowest drift times due to their high charge; [(PAA-VG2)1 +2H] +2 ions with one block show intermediate drift velocities and drift times, whereas the [PAA+K] + and [PAA+Na+K] +2 components are the slowest ions because they carry one charge only or do not include the peptide which would facilitate folding and faster drifting motion. 1030.5444 [(PAA₁₀-VG₂)₂+4H]+⁴ [(PAA₁₀-VG₂)₁+2H]+² 3.88 5.42 6.95 m/z 1030 [(PAA₆+K]+& [(PAA₂₄+Na+K]+² 1030 1032 m/z 0.00 2.50 5.00 7.50 10.00 drift time (ms) 1102.5675 [(PAA₁₂-VG₂)₁+2H]+² m/z 1102 [(PAA₁₂-VG₂)₂+4H]+⁴ 5.96 7.70 4.06 7.13 [(PAA₁₁+K]+& [(PAA₂₆+Na+K]+² 1102 1104 m/z 0.00 2.50 5.00 7.50 10.00 drift time (ms) Figure 4.32 IM-MS chromatogram of the ions at m/z 1030 and m/z 1102 from PAA-VG2. The insets show the mass spectra extracted from the center components. 102
IM-MS analysis of triply charged PAA-VG2 oligomers also indicates the presence of overlapping isobars, as exemplified in Figure 4.33 for [(PAA15-VG2)1 +3H] +3 (m/z 807) and [(PAA17-VG2)1 +3H] +3 (m/z 831). Here, the superimposed component is unreacted, longer chain PAA with overall +3 charges (Figure 4.33). The longer drift time of the unreacted PAA is attributed to its longer chains (compared to the PAA block in PAA-VG2) and the lack of the peptide block which would promote folding by hydrogen bending between the polyacrylate COOH groups and the VG2 amide groups. 2.98 [(PAA₁₅-VG₂)₁+3H]+³ m/z 807 6.05 [PAA₂₉+H+Na+K]+³ 808.4122 808.7261 832.7314 832.4130 831.0657 832.0734 831.7445 807.0636 807.3981 807.7430 808.0776 831.4050 807 809 m/z 2.50 5.00 7.50 10.00 drift time (ms) [(PAA₁₆-VG₂)₁+3H]+³ 3.07 6.23 [PAA₃₀+H+Na+K]+³ m/z 831 1102 1104 m/z 2.50 5.00 7.50 10.00 drift time (ms) Figure 4.33 IM-MS chromatogram of the ions at m/z 807 and m/z 831 from PAA-VG2. The insets show the mass spectra extracted from the faster drifting components. 103
Table 4.4 Experimental collision cross-sections of twelve doubly and triply protonated PAAn-VG2 oligomers, derived using the calibration plot of Figure 4.30. z n m n-mer (Da) m/z t D (ms) t D` (ms) Reduced mass Ω' (Ų) Ω (Ų) 2 7 1841.93 921.971 4.75 4.7790 5.2527 838.490 336 2 8 1912.76 957.387 4.89 4.9195 5.2541 897.708 342 2 10 2057.84 1029.926 5.47 5.4957 5.2568 954.163 363 2 12 2201.88 1101.950 5.96 5.9918 5.2591 1000.675 381 2 14 2346.13 1174.072 6.28 6.3129 5.2611 1029.858 391 2 15 2417.94 1209.976 6.39 6.4184 5.2621 1039.302 395 2 16 2490.88 1246.447 6.93 6.9639 5.2629 1087.055 413 3 10 2058.06 687.027 2.61 2.6348 5.2568 636.500 363 3 12 2201.84 734.954 2.69 2.7157 5.2591 647.190 369 3 14 2346.04 783.022 2.82 2.8466 5.2611 664.184 379 3 15 2417.89 806.971 3.03 3.0570 5.2621 690.788 394 3 16 2490.08 831.034 3.08 3.1074 5.2629 697.040 397 Table 4.4 lists the drift times of twelve doubly and triply protonated PAA-VG2 oligomers with one constituent block. The narrow drift time distributions of these ions point to a single architecture. The drift times are lower for the triply charged ions because they move faster than the doubly charged ions of PAA-VG2 inside triwave IM region. Table 4.4 also includes the collision cross-sections obtained from the measured drift times by using the calibration curve established with polyalanine oligomers of known CCS. The CCSs for doubly charged ions vary between 336 Ų and 413 Ų for oligomers 104
CCS CCS with 7-16 PAA repeat units. For the triply charged ions, the CCS range is 363-397 Ų for oligomers with 10-16 PAA repeat units. In general, the drift times and experimental CCSs of PAA-VG2 oligomers are lower comparing with the drift times and experimental CCSs of PtBA-VG2 oligomers with the same number of acrylate repeat units (Figure 4.34), because PAA lacks the bulky t-butyl substituents and enables hydrogen bonding that generates folded, compact structures in PAA-VG2 hybrid material (vide infra). (a) PtBA-VG₂ and PAA-VG₂ +2 ions 500 450 400 PtBA-VG2 PAA-VG2 350 300 800 900 1000 1100 1200 1300 1400 m/z (b) PtBA-VG₂ and PAA-VG₂ +3 ions 480 460 440 420 400 380 360 340 320 300 600 650 700 750 800 850 900 950 m/z PtBA-VG2 PAA-VG2 Figure 4.34 Plot of experimental collision cross-section vs. m/z for PtBA-VG2 and PAA- VG2 oligomers with (a) +2 and (b) +3 protonated charges. 105
4.7.2 Molecular Modeling The structure of the hybrid materials could be linear or cyclic depending on whether intramolecular cycloaddition between the chain ends occurs. To answer this question, computational modeling was used. Energy minimization of PtBA-VG2 and PAA-VG2 oligomers with different numbers of acrylate repeat units and linear or cyclic structures was preformed by molecular mechanics/dynamics simulations using the Anneal and Geometry Optimization tasks in the Forcite module of the Materials Studio (version 7.0) program (Accelrys Software, Inc.). The number of annealing cycles was 50 for each initially energy-minimized structure. The initial and midcycle temperatures were 50 and 1400 K, respectively, with 20 heating ramps per cycle, 1000 dynamics steps per ramp, and one dynamics step per femtosecond. In MOBCAL algorithm was used to calculate collision cross-sections by the projection approximation (PA), exact hard sphere scattering (EHSS) and trajectory(tj) methods for each of the 50 simulated structures (from the Materials Studio program) of cyclic and linear PtBA-VG2 and PAA-VG2. The cross-sections obtained by the projection approximation method are plotted against the corresponding relative energies (from the Materials Studio program) in Figures 4.35 and 4.36 to illustrate the energetic and structural differences between cyclic and linear architectures of PAA-VG2 and PtBA- VG2. 106
Cross-section (Ų) Cross-section (Ų) (a) 600 PtBA₆-VG₂ 550 500 450 400 350 300 0 10 20 30 40 50 60 70 80 90 Relative Potential Energy (kcal/mol) (b) 600 PtBA₇-VG₂ 550 500 450 400 350 300 0 20 40 60 80 100 120 Relative Potential Energy (kcal/mol) Figure 4.35 Plot of calculated collision cross-section (PA method) vs. relative energy for 50 energy-minimized structures of a) [PtBA₆-VG2] +2 (m/z 1054.1), and b) [PtBA7- VG2] +2 m/z 1118.2) with linear architecture (orange circle) or cyclic architecture (blue triangle). On average, the cyclic architectures are more stable (by ~ 2.9 kcal/mol). Representative linear ans cyclic structures are shown in the lower right and upper left corners, respectively. 107
550 500 Cross-section (Ų) Cross-section (Ų) (a) PAA₇-VG₂ 450 400 350 300 0 10 20 30 40 50 60 70 Relative Potential Energy (kcallmol) (b) 600 PAA₁₀-VG₂ 550 500 450 400 350 300 0 10 20 30 40 50 60 70 80 90 Relative Potential Energy (kcal/mol) Figure 4.36 Plot of calculated collision cross-section (PA method) vs. relative energy for 50 energy-minimized structures of a) [PAA7-VG2] +2 (m/z 921.97), and b) [PAA10-VG2] +2 (m/z 1029) with linear architecture (orange circle) or cyclic architecture (blue triangle). On average, the cyclic architectures are more stable (by ~ 4 kcal/mol). Representative linear ans cyclic structures are shown in the lower right and upper left corners, respectively. 108
Table 4.5 compares experimental and calculated collision cross-sections of several doubly and triply protonated PtBA-VG2 oligomers. The experimental CCSs were obtained from Table 4.3. The calculated CCSs were obtained from the energy-minimized structures using the trajectory (TJ), projection approximation (PA), and exact hard sphere scattering (EHSS) methods available in the MOBCAL program. The projection approximation (PA) estimates the average geometric cross-section by disregarding the scattering process between the ion and the buffer gas. The exact hard sphere scattering (EHSS) method accounts for the scattering process by considering the momentum transfer occurring in the ion/buffer gas collision. Long-range interactions between the ion and buffer gas atoms are neglected in the PA and EHSS methods. In the trajectory (TJ) method, the effective ion potential is calculated by summing the Lennard-Jones potential of each atom in the ion and adding long-range ion-induced dipole interactions (with the charge delocalized over all atoms), and this potential is then used to determine the scattering angles of the buffer gas atoms. 143,144 The TJ method considers scattering phenomena, ion-buffer gas interactions, and multiple collisions between ion and buffer gas and, hence, provides the most reliable results, especially for larger, non-spherical ions, but is time consuming and expensive. For smaller ions with fairly globular structures, the PA method has been shown to provide adequate CCSs. 64 Figure 4.37 shows plots that compare the calculated collision cross-sections (PA method) of linear and cyclic PtBA-VG2 oligomers in charge states +2 or +3 with the corresponding experimental collision cross-sections. The experimental CCSs match within experimental error (~4 %), the CCSs calculated for the cyclic structures indicating 109
that all possible 1+3 cycloadditions have taken place and that the hybrid material contains solely triazole functionalities and no free azide and alkyne end groups. Table 4.5 Collision cross-sections of doubly and triply protonated of PtBA-VG2 oligomers. The experimental CCSs were obtained from Table 4.3. The calculated values are the average CCSs of the 50 energy-minimized structures, obtained using the projection approximation (PA), exact hard sphere scattering (EHSS), and trajectory (TJ) methods of the MOBCAL program. n z m/z t D (ms) CCS (exp) (Ų) CCS (calcd) (Ų) Architecture PA EHSS TJ 4 2 926.05 5.50 365 Cyclic 367 430 418 Linear 388 455 442 6 2 1054.19 6.41 396 Cyclic 396 467 456 Linear 428 503 490 7 2 1118.25 7.05 417 Cyclic 411 485 474 Linear 444 525 510 8 2 1182.28 7.70 438 Cyclic 430 510 498 Linear 458 542 527 10 2 1310.36 8.72 468 Cyclic 464 553 539 Linear 499 591 576 11 2 1374.42 Cyclic 483 576 562 Linear 520 618 602 12 2 1438.46 Cyclic 497 594 581 Linear 524 625 609 5 a 3 732.44 3.08 398 Cyclic 404 474 470 Linear 431 508 501 6 a 3 775.17 3.33 415 Cyclic 416 492 487 Linear 449 531 524 7 a 3 817.84 3.59 432 Cyclic 439 521 516 Linear 465 549 541 9 a 3 903.23 4.00 458 Cyclic 469 559 554 Linear 501 599 588 a the triply protonated oligomers also contain 3 hydrolyzed PAA repeat units. 110
CCS CCS (a) PtBA n -VG₂ (+2 charge) 600 550 500 450 4 n= 6 7 8 10 11 12 PtBA-VG2 Cyclic calc PtBA-VG2 Linear calc PtBA-VG2 exp 400 350 300 900 1000 1100 1200 1300 1400 1500 m/z (b) PtBA n PAA₃-VG₂ (+3 charge) 600 550 500 450 400 n= 5 6 7 9 PtBA-VG2 Cyclic calc PtBA-VG2 Linear calc PtBA-VG2 exp 350 300 700 750 800 850 900 950 m/z Figure 4.37 Plot of calculated collision cross-sections (PA method) for PtBA-VG2 oligomers with linear and cyclic structures and (a) +2 or (b) +3 charges, and experimental collision cross-sections of the same oligomers vs. m/z ratio. 111
Table 4. 6 Collision cross-sections of doubly and triply protonated of PAA-VG2 oligomers. The experimental CCSs were taken from Table 4.4. The calculated values are the average CCSs of the 50 energy-minimized structures, obtained using projection approximation (PA), exact hard sphere scattering (EHSS), and trajectory (TJ) methods of the MOBCAL program n z m/z t D (ms) CCS (exp) (Ų) CCS (calcd) (Ų) Architecture PA EHSS TJ 7 2 921.97 4.75 336 Cyclic 352 411 403 Linear 376 439 427 8 2 957.39 4.89 342 Cyclic 366 429 420 Linear 395 460 448 10 2 1029.93 5.47 363 Cyclic 389 458 447 Linear 413 484 472 12 2 1101.95 5.96 381 Cyclic 390 459 450 Linear 432 508 495 14 2 1174.07 6.28 391 Cyclic 416 492 483 Linear 464 544 530 15 2 1209.98 6.39 395 Cyclic 426 504 484 Linear 459 541 529 16 2 1246.45 6.93 413 Cyclic 436 517 506 Linear 471 556 543 10 3 687.03 2.61 363 Cyclic 386 453 447 Linear 417 488 477 12 3 734.95 2.69 369 Cyclic 408 480 475 Linear 445 521 510 14 3 783.02 2.82 379 Cyclic 413 487 481 Linear 457 537 527 15 3 806.97 3.03 394 Cyclic 430 508 502 Linear 474 557 542 16 3 831.03 3.08 397 Cyclic 437 518 511 Linear 483 569 558 Table 4.6 lists the collision cross-sections of doubly and triply protonated PAA- VG2 oligomers with linear or cyclic architecture, calculated from simulated structures by using the PA method. Figure 4.38 shows the same result in form of a graph that includes the corresponding experimental collision cross-sections. 112
CCS (a) CCS PAA n -VG₂ (+2 charge) 600 550 n= 500 14 15 16 12 450 10 7 8 400 350 300 250 200 900 1000 1100 1200 1300 m/z PAA-VG2 Cyclic PAA-VG2 Linear PAA-VG2 exp (b) PAA n -VG₂ (+3 charge) 600 550 500 450 n= 10 12 14 15 16 PAA-VG2 Cyclic PAA-VG2 LINEAR PAA-VG2 EXP 400 350 300 650 700 750 800 850 m/z Figure 4.38 Plot of calculated collision cross-sections (PA method) for PAA-VG2 oligomers with linear and cyclic structures and (a) +2 or (b) +3 charges, and experimental collision cross-sections of the same ions vs. m/z ratio. As with PtBA-VG2, the cyclic PAA-VG2 architectures are more compact and have smaller collision cross-sections than their linear analogs. In contrast to PtBA-VG2, however, the experimental CCSs do not match those of the macrocycles, but are even smaller. This increase in compactness (decrease in CCS) probably results from the 113
extensive hydrogen bond network formed with the COOH pendants of PAA chains and the VG2 amide groups. It is likely that the molecular mechanics/ dynamics simulations miss some of the most compact PAA-VG2 conformations, leading to overestimated CCS values. 4.8 Conclusions The two hybrid metarials in this study are cyclic and they have at least two blocks of polymer-peptide block. The results presented in this chapter showed how top-down MS, involving tandem MS (MS n ) and ion mobility mass spectrometry (IM MS), can be used to characterize a derivatized synthetic peptide (complete end group and sequence analysis), a polymer, and the corresponding polymer peptide hybrid copolymer. Topdown MS is a new approach suitable for the analysis of complex materials that can not be obtained in the high purity needed for structural characterization by other spectroscopic methods such as XRD and NMR. Top-down MS is fast, requires very little material, and can reveal information about both the composition as well as the architecture microstructure of the materials under study. 114
CHAPTER V MASS SPECTROMETRY CHARACTERIZATION OF BIOACTIVE PEPTIDE - SYNTHETIC POLYMER CONJUGATES 5.1 Background Bone remodeling involves the removal and replacement of bone that has been damaged by diseases or sports activities. 145 Copper and bronze materials were used to repair damaged bone tissues in the middle of the nineteenth century, but these materials corrode in human bodily fluids, which can cause infections to the patient. In the late nineteenth and early twentieth century, new materials like gold, glass, and alloys were used to solve bone replacement issues. During the second half of the twentieth century, ceramic materials, which have good chemical, physical and mechanical properties, were utilized to rebuild and replace tissues for dental and orthopedic implants. They were not harmful to the body and exhibited corrosion resistance. 146 More recently, biomedical polymers like poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), 147 polyamide (PA), poly(ε-caprolactone) (PCL), and poly(lactic-co-glycolic acid) (PLGA) have been utilized in the manufacture of bone implants and for tissue engineering. While these polymers can be used to form bone 115
tissue, the bone cannot spontaneously bond with the polymer surface because such materials are not bioactive. 148 Titanium and its compounds have also been utilized for several decades in the manufacture of bone implants but again they are not bioactive materials. 149 To solve this problem, bioactive materials had been used to coat the surfaces of implants made of steel 150 and titanium. 149, 151 Bioactive materials should be biocompatible, hypoallergenic, noninflammatory, and nontoxic and they should prevent corrosion of the covered metal and the release of metal ions into the implant environment. 151 Bone and teeth tissues consist of cells, natural polymer (for example, polysaccharides and collagen), and mineral components like apatites which have the general formula M10 (XO4)6 (Y)2, where M is a bivalent metal cation, XO4 is a trivalent anion, and Y is a monovalent anion. The apatite s name is based on the identity of the M, X, and Y atoms. 152, 153 Hydroxyapatite (HA) is a calcium phosphate bioactive material that promotes the adhesion, growth, and osteogenic differentiation of bone-making cells (osteoblasts); 153 it has been widely in use since the early 1980s because of its excellent connectivity with bone tissues. 154 Several synthetic methods have been described for HA whose chemical formula is Ca10(PO4)6(OH)2 155-160 HA is biocompatible, osteoconductive, biodegradable, and able to connect with biomolecules without affecting their biological function; due to these properties HA has been used in bone remodeling processes. 145 On the other hand, HA has low break strength and has no antibacterial properties to prevent implant-associated infection. 161-163 Matthew L. Becker and coworkers found that the peptide GGGSVSVGMKPSPRP (called " HA peptide") can strongly bind to crystalline hydroxyapatite, or the HA-containing portions of a human 116
tooth and thus can be used as a linker to attach other bioactive molecules to this mineral. 164 Proteins are also used in bone remodeling applications and are typically added to the bioactive materials 165,167 or immobilized onto a polymer like polycaprolactone (scaffold). 168 Bone morphogenic proteins (BMPs), which belong to the transforming growth factor beta (TGFb) family of growth factors, play an important role in promoting bone formation, 168-171 osteoblast recruitment, and angiogenesis. 172 The BMP-2 peptide is a receptor binding site on BMP-2 proteins, and it plays an important role in stimulating bone cells to induce calcification. The sequence of BMP-2 peptide is KIPKASSVPTELSAISTLYL and contains amino acid residues 73-92 from the knuckle epitope of BMP-2 protein. 173 Recently, more than several research groups have used both a recombinant BMP and hydroxy apatite in bone regeneration applications. 98,174-176 At the University of Akron, Matthew L. Becker and coworkers synthesized a new hybrid material [BMP- 2(HA)2], which was developed for use as a tissue engineering substrate in bone healing applications. This polymer-peptide based hybrid material has a dendridic structure containing two polyethylene glycol (PEG) branches terminated by a bioactive hydroxyapatite binding peptide (HA), and a focal point substituted with a different bioactive peptide (BMP-2) that mimics the bone morphogenic protein-2. 98 In this study, [BMP-2(HA)2] is characterized by multiple mass spectrometry methods, including MALDI-ToF-MS, ESI-QIT-MS, and ESI-Q/ToF-MS coupled with ion mobility spectrometry. These methodologies would be equally applicable to biologics and biosimilars, many of which are composed of protein (peptide)/polymer conjugates smiliar 117
as [BMP-2(HA)2]. 5.2 Sample Preparation and Instruments Used The samples analyzed were prepared in Dr. Becker s group (University of Akron), and encompass the BMP-2 peptide, HA peptide, and the hybrid material [BMP- 2-(HA)₂]. 98 5.2.1 Sample Preparation for MALDI-ToF/ToF-MS MS and MS² experiments were performed on a Bruker UltraFlex III MALDI tandem time-of-flight (ToF/ToF) mass spectrometer operated in positive mode (Bruker Daltonics, Billerica, MA, USA) equipped with a Nd:YAG laser emitting at 355 nm. For the BMP-2 and HA peptides, α-cyano-4-hydroxycinnamic acid (CHCA) was used as matrix. A solution of CHCA matrix was prepared in THF at a concentration of 20 mg/ml and was employed to make the top and bottom layers of the samples deposited onto the MALDI target. Solutions of the peptides were prepared in H₂O at a concentration of 20 mg/ml and were used to add the center layer of the samples (sandwich method). This sample preparation procedure led to the formation of abundant [BMP-2 + H]+ and [HA + H]+ ions from both peptides. For the [BMP-2-(HA)₂] hybrid material, sinapinic acid (SA) served as matrix. A solution of SA (in ACN: H2O) (70:30, v/v %) was prepared at a concentration of 20 mg/ml; it was used to make the top and bottom layers of the sample subjected to MALDI. A [BMP-2-(HA)₂] solution (10 mg/ml) was prepared in H₂O. The two 118
solutions were combined onto the MALDI target via the sandwich method. This sample preparation protocol led to the formation of singly and doubly protonated ions. 5.2.2 Sample Preparation for ESI-QIT-MS MS and MS² experiments were performed on a quadrupole ion trap (QIT) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with an electrospray ionization (ESI) source operated in positive ion mode. The peptide sample solutions were injected into the instrument by direct infusion at a flow rate of 3 μl/min. the ESI needle was gronnded and the entrance into the vacuum system of the QIT was kept at 3.5 kv. The nebulizer gas pressure held at 10 psi, and the drying gas flow rate and temperature at 8 L/min and 300 C, respectively. The peptides were dissolved in H₂O at a concentration of 0.01 mg/ml and 10% MeOH (v %) was added to this solution to help the spray process. CAD experiments were carried out by using helium as collision gas and setting the excitation amplitude value between 0.18 and 0.90 (arbitrary units), depending on the precursor ion isolated. For the experiments involving ETD, the anion reagent species were generated in a negative chemical ionization (nci) source, which was tuned to maximize the generation and transmission of the ETD reagent ions (fluoranthene radical anions) as follows: reagent ion ICC 100000, ionization energy 70 ev, emission current 2.0 μa, reactant remove cut off m/z 210, and methane as buffer gas. After the accumulation of both precursor ions and ETD reagent radical anions inside the ion trap, the reaction time was set in the range between 100-200 ms, depending on the peptide investigated and the precursor ion selected. 119
5.2.3 Sample Preparation for ESI-Q/ToF-MS MS and MS² experiments were performed on a Waters Synapt HDMS quadrupole/time-of-flight (Q/ToF) mass spectrometer (Waters, Beverly, MA, USA), equipped with an electrospray ionization (ESI) source. The sample solution was introduced to the ESI source by direct infusion. The instrument was operated in positive ion mode with a capillary voltage of 3.15 kv, cone voltage of 35 V, sampling cone voltage of 3.2 V, source temperature of 80 C, and desolvation temperature of 150º C. The BMP-2 peptide was dissolved in H₂O at a concentration of 0.05 mg/ml; 10% of MeOH and 1% formic acid (both v/v %) were added to this solution to help in the evaporation and ionization of the sample. The HA peptide was dissolved in H₂O at a concentration of 0.01 mg/ml and 10% of MeOH (v/v %) was added to this solution. The [BMP-2-(HA)₂] sample was dissolved in H₂O at a concentration of 0.01 mg/ml and 30% of MeOH plus 0.5% formic acid (both v/v %) were added to maximize ion formation upon ESI. Ion mobility separation was achieved by tuning the wave height and wave velocity in the triwave ion mobility spectrometry (IMS) device to 8 V and 300 m/s (for the peptides), or 8 V and 250 m/s (for the [BMP-2-(HA)₂] hybrid material), respectively; the wave height and wave velocity in the trap cell were set to 0.5 V and 300 m/s, respectively; the wave height and wave velocity in the transfer cell were set to 0.2 V and 248 m/s, respectively. The nitrogen gas (drift gas) flow rate was 22 L/h. Tandem mass spectrometry experiments were performed in the transfer cell, which is located after the ion mobility chamber, using argon as collision gas. 120
5.3 Characterization of BMP-2 Peptide This chapter reports the procedure developed for the analysis of BMP-2 peptide. BMP-2 is a hydrophilic peptide with twenty amino acid residues, viz. four serine units; three leucine units; two units of lysine, threonine, proline, alanine, and isoleucine; and one unit of glutamic acid, tyrosine and valine. The peptide was prepared via solid state synthesis on a microwave peptide synthesizer. 98 The sequence expected from the synthetic process is KIPKASSVPTELSAISTLYL. 177 The protonated form of this peptide has a monoisotopic mass of 2118.2056 Da. [BMP-2+Na]+ 2140.479 [BMP-2+H]+ 2118.500 [BMP-2+K]+ 2156.475 1000 1200 1400 1600 1800 2000 2200 m/z Figure 5.1 MALDI-MS spectrum of BMP-2 peptide dissolved in H2O at 20 mg/ml. CHCA (20 mg/ml) served as matrix and the sandwich method was used. 121
5.3.1 Characterization of BMP-2 by MALDI-ToF/ToF-MS Figure 5.1 shows the MALDI-MS spectrum of BMP-2 peptide dissolved in H₂O, using CHCA as matrix. The most abundant peaks arise from the protonated ion at m/z 2118.500 ([BMP-2+H]+) and the quasi-molecular ions at m/z 2140.479 ([BMP-2+Na]+) and m/z 2156.475 ([BMP-2+K]+). The BMP-2 peptide sequence was examined by tandem mass spectrometry. The MALDI-MS 2 spectrum of protonated BMP-2, i.e. [BMP- 2+H]+ (m/z 2118.50), is shown in Figure 5.2. The fragments observed confirm the sequence KIPKASSVPTELSAISTLYL, with a lysine residue at the N-terminus and a leucine residue at the C-terminus. The most abundance fragments in the Figure belong to the N-terminal bn+1 fragment family, formed by amide bond cleavages, and the complementary C-terminal yn+1 fragment family, formed of by the same type of bond cleavages. The intensities of y 18 and b 8 are high because of enhanced amide bond cleavage at the N-terminal side of proline, which is facilitated by the high proton affinity and secondary amine structure of this amino acid. A complete series of N-terminal bn+1 sequence ions (from b18 until b3) can be seen due to the presence of highly basic amino acids at and near the N-terminus, in particular lysine (residues 1 and 4) and proline (residue 3). 5.3.2 Characterization of BMP-2 by ESI-QIT-MS and ESI-Q/ToF-MS The best signal intensities on the QIT mass spectrometer were achieved with aqueous solutions of the peptide containing 10% MeOH (Figure 5.3). The BMP-2 peptide forms multiply charged ions (+2, +3 and +4) under ESI conditions. The most 122
181.428 384.652 625.973 1611.117 1712.105 129.3382 PT-H₂O 328.556 441.658 471.736 685.027 898.256 552.832 1011.406 1339.794 2027.555 PTEL 784.187 1139.535 1252.707 1410.858 1825.308 226.494 713.047 1988.431 1523.995 1878.307 570.856 y₁₈ b 7 b8 b₁₀ b₁₂ b₁₈ b₃ b₄ b₅ b₆ b₉ b₁₁ b₁₃ b₁₄ b₁₅ b₁₆ b₁₇ b₁₉ 812.204 b₈ KASSVP 2118.495 y₁₈+1 K AAS-H₂O b₄ b₃ b₅ b₆ a₇ b₇ a₈ b₉ b₁₀ b₁₁ b₁₂ b₁₃ b₁₄ b₁₅ b₁₆ b₁₇ b₁₈ b₁₉ a₅ a₁₂ a₁₄ 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z Figure 5.2 MALDI-MS 2 spectrum of protonated BMP-2 (m/z 2118.500) acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum. All b and a fragments in this Figure are of the type bn+1, yn+1 and an+1 (radical ions). abundant peaks in Figure 5.3 arise from the triply charged ions, which include the protonated ion at m/z 706.8 ([BMP-2+3H]+³) and the quasi-molecular ions at m/z 714.1 ([BMP-2+2H+Na]+³), m/z 719.4 ([BMP-2+2H+K]+³), m/z 721.4 ([BMP-2+2Na+H]+³), m/z 726.7 ([BMP-2+H+Na+K]+³), and m/z 728.7 ([BMP-2+3Na]+³). The protonated triply charged ion at m/z 706.8 ([BMP-2+3H]+³) is the most abundant ion in the triply charged ion group. The intensities of the quadruply charged ions, which include m/z 535.8 ([BMP-2+3H+Na]+⁴), m/z 539.8 ([BMP-2+3H+K]+⁴), m/z 541.3 ([BMP-2+2H+2Na]+⁴), 123
663.4 848.1 1060.2 539.8 m/z 545.5 ([BMP-2+2H+Na+K]+⁴), and m/z 546.8 ([BMP-2+H+3Na]+⁴) are lower compared with those of the triply charged ions but higher than those of the doubly charged ions. The most abundance ion in the +4 charge state is m/z 539.8 ([BMP- 2+3H+K]+⁴). The doubly charged ions include the protonated ion at m/z 1059.6 ([BMP- 2+2H]+²) and the quasi-molecular ion at m/z 1059.6 ([BMP-2+H+Na]+²). [BMP-2]+³ 707.0 [BMP-2 +2H +Na]+³ 714.1 [BMP-2 +2H +K]+³ 719.4 726.7 728.7 1070.6 1071.1 1059.6 545.5 546.8 535.8 541.3 (B) [BMP-2 +3H +K]+⁴ 539.8 [BMP-2 +3H +Na]+⁴ [BMP-2 +2Na +2H]+⁴ [BMP-2 +2Na +K+H]+⁴ [BMP-2 +3Na +H]+⁴ 534 536 538 540 542 544 546 m/z [BMP-2 +3H]+³ 706.8 (C) 721.4 [BMP-2 +3Na ]+³ [BMP-2 +2Na+H]+³ [BMP-2 +H+K+Na ]+³ 705 710 715 720 725 730 m/z [BMP-2+2H]+² 1060.2 1060.7 (D) [BMP-2 +Na +H]+² [BMP-2]+⁴ (A) [BMP-2]+² 1060 1064 1066 1058 1068 1070 1072 1074 1062 m/z 250 500 750 1000 1250 1500 1750 2000 2250 m/z Figure 5.3 A) ESI-MS spectrum of BMP-2 peptide dissolved in H2O at 0.01 mg/ml + 10 % (v %) of MeOH, acquired with the QIT mass spectrometer. B-D) Expanded views of the m/z regions of B) quadruply charged ions (green box), C) triply charged ions (red box), and D) doubly charged ions (blue box) of BMP-2 peptide. 124
y₁₈ b 7 b8 b₁₀ b₁₂ b₁₈ b₃ b₄ b₅ b₆ b₉ b₁₁ b₁₃ b₁₄ b₁₅ b₁₆ b₁₇ b₁₉ [BMP-2+2H]+² 1061.0 PTEL 441.1 PKASSV 570.3 b₇ 712.5 b₈ 811.6 (y₁₈+1 )+² b₁₈+² b₁₉+² 913.1 994.6 PTELSAISTLY-H₂O PTELSAISTLY PKASSVPTELSAI or IPKASSVPTELSA b₁₁ 1138.6 y₁₂ b₁₄+1 1307.7 1410.8 b₁₃ b₁₅+1 1523.9 b₁₆+1 PKASSVPTELSAISTLY y₁₈ 400 600 800 1000 1200 1400 1600 1800 2000 m/z Figure 5.4 ESI-CAD mass spectrum of the doubly protonated BMP-2 peptide, [BMP- 2+2H] +2 (m/z 1059.6), acquired on the ESI-QIT mass spectrometer. The CAD spectrum of the doubly protonated peptide, [BMP-2+2H]+² at m/z 1059.6 (Figure 5.4), shows mostly yn and a few bn fragments arising from peptide bond cleavages, as was the case upon MALDI-MS 2 of [BMP-2 +H] + (Figure 5.2). As mentioned before, the predominance of b-type ions is due to the presence of highly basic amino acids at and near the N-terminus; however, the bn (or bn+1) series is less complete than in the MALDI-MS 2 spectrum. The relative intensities of b8, y12, and (y18+1) +2, which 125
are formed by cleavages of peptide bonds at the N-terminal side of Pro, are particularly high. Fragments with 16 or less residues are singly charged, whereas larger fragments are observed with +2 charges. c₇ c₉ c₁₁ z₁₉ z₁₈ c₆ c₁₀ c₁₂ c₁₃ c₁₄ c₁₅ c₁₆ c₁₇ c₁₈ c₁₉ [BMP-2+2H]+ [BMP-2+2H]+² 1059.6 2119.1 c₁₃-1 ELSA 401.0 c₈-1 c₇-1 728.3 641.3 c₉-1 924.5 c₁₀-1 1025.8 c₁₁-1 1154.7 c₁₂-1 1354.8 c₁₄-1 1267.8 1425.9 c₁₅-1 1538.8 c₁₆-1 1625.9 c₁₈-1 c₁₇-1 z₁₇ 1727.1 1764.0 1840.1 z₁₉ c₁₉ 2004.1 1974.1 400 600 800 1000 1400 1600 1800 2000 m/z Figure 5.5 ESI-ETD mass spectrum of the doubly protonated BMP-2 peptide, [BMP- 2+2H] +2 (m/z 1059.6), acquired on the ESI-QIT mass spectrometer. The ETD spectrum of the same ion, i.e. m/z 1059.6, shows a cn fragment ion series (Figure 5.5) that is largely contiguous (from c9 to c19) and covers 60% of the peptide sequence. All fragments agree well with the BMP-2 sequence. Interestingly, the 126
241.9 406.1 STLY 498.2 603.0 1168.6 663.4 811.5 y₁₈-h₂o+² 1307.7 294.9 PTELSAISTL-H₂O+² 570.3 KASSVP b₁₈-h₂o+³ [BMP-2]+³ 762.5 PKASSVPTELSA majority of cn fragments are observed as cn-1 radical ions. Further, fragments from cleavage of the proline N-C α bonds (c8 or c8-1) are not detected, as also found for other multiply protonated peptides. 57,133 in addition to c-type fragments, ETD of doubly protonated BMP-2 also produces one z-type fragment (z19) as well as the change-reduced ion [BMP-2+2H] + at m/z 2119.1. Both ESI-QIT spectra (CAD and ETD) suggest that both protons are attached on the N-terminal side of the peptide. y₁₈ b 7 b8 b₁₀ b₁₂ b₁₈ b₃ b₄ b₅ b₆ b₉ b₁₁ b₁₃ b₁₄ b₁₅ b₁₆ b₁₇ b₁₉ b₁₈+² 913.0 b₁₄-h₂o+² b₁₉+² 994.5 b₁₆+² b₂ y₂ b₈+² b₁₅+² b₁₇+²-nh₃ b₁₉+³ b₈ y₁₂ y₁₈+² b₁₇+² 200 400 600 800 1000 1200 1400 m/z Figure 5.6 ESI-CAD mass spectrum of the triply protonated BMP-2 peptide, [BMP- 2+3H] +3 (m/z 706.8), acquired on the ESI-QIT mass spectrometer. 127
The CAD spectrum of the triply protonated peptide, viz. [BMP-2+3H] +³ at m/z 706.8 Da (Figure 5.6), shows an incomplete series of yn and bn fragments in different charges. The significant relative abundances of the b2/y18 +2, b8 +2 /y12, and b8/y12 pairs corroborate once more the preference for selective peptide bond cleavage N-terminal to proline. The high relative abundances of b18 +2 /y2 and b19 +2 are also noteworthy; they indicate facile cleavage of the C-terminally last and penultimate amide bonds in the triply charged BMP-2 peptide. c₇ c₉ c₁₁ z₁₉ z₁₈ c₆ c₁₀ c₁₂ c₁₃ c₁₄ c₁₅ c₁₆ c₁₇ c₁₈ c₁₉ [BMP-2+3H]+³ 706.6 [BMP-2+2H]+² 1059.6 b₁₄ 1409.9 355.9 c₃-1 484.2 555.2 641.3 1155.6 1268.8 1354.7 1539.6 1626.8 1728.1 1841.1 2004.1 2101.1 c₆-1 921.0 864.5 813.4 1002.5 KASSVPTELSAIST-H₂O c₄ c₅ c₁₅+²c₁₇+² c₁₈+² c₁₉+² c₁₁ c₁₂ c₁₅ c₁₆ c₁₇ c₁₈ [BMP-2+H]+ c₁₉ 2118.1 [BMP-2+H-NH₃]+ 250 500 750 1000 1250 1500 1750 2000 2250 m/z Figure 5.7 ESI-ETD mass spectrum of the triply protonated BMP-2 peptide, [BMP- 2+3H] +3 (m/z 706.6), acquired on the ESI-QIT mass spectrometer. 128
218.7 356.0 642.3 1021.4 1428.6 1515.8 1673.8 1602.8 1800.9 2041.9 2155.9 ASS-28 539.2 484.2 837.3 *z₁₃ *a₁₆-nh₃ *z₁₆ *z₁₇ *z₁₆+² *a₁₅-nh₃ [BMP-2+K+3H]+⁴ 1078.5 681.3 * c₁₉+³ The ETD spectrum of the same ion (m/z 706.6) is depicted in Figure 5.7. It contains partially contiguous series of singly and doubly charged c-type ions, which are in full agreement with the BMP-2 peptide sequence. Interestingly, the majority of the c- type fragments are closed-shell cn ions; in contrast, the doubly protonated peptide produced predominantly cn-1 radical ions upon ETD (vide supra). Hence, the precursor ion charge appears to affect the radical transfers occurring during ETD between the emergent fragments and/or between the reduced precursor and the ETD reagent. [BMP-2+3H+K] +³ 719.6 c₁₀+² 513.6 c₃ c₄ c₆ 901.4 *z₁₇+² [BMP-2+H+K]+² c₁₉+² 1006.4 z₁₉+² *a₁₁-nh₃ 1131.4 *z₁₉ [BMP-2+K]+ c₇ c₁₁-1 1154.6 250 500 750 1000 1250 1500 1750 2000 m/z Figure 5.8 ESI-ETD mass spectrum of the quadruply charged BMP-2 peptide, [BMP- 2+3H+K] + ⁴ (m/z 539.8), acquired on the ESI-QIT mass spectrometer. An asterisk indicates that the ion contains K +. 129
The ETD spectrum of the quadruply charged peptide with the composition [BMP- 2+3H+K]+⁴ (m/z 539.8) Figure 5.8, shows cn, zn, and an fragments in different charge states, some with and some without the potassium ion. Generally, the zn ions observed contain 13 residues, wheres the cn ions are relatively small ( 11 residues). [BMP-2+4H+K]+³ 719.9 c₁₁+² y₁₈+³ 578.2 638.9 c₁₃+² c₁₄+² 713.5 y₁₆+² 845.3 y₁₈+² c₁₆+² 957.9 c₁₅+² *z₉ c₁₇+² 788.9 832.3 883.2 1002.6 c₁₁ 1155.5 200 400 600 800 1000 1200 1400 m/z Figure 5.9 ESI-MS³ mass spectrum acquired on the ESI-QIT mass spectrometer by CAD of [BMP-2+3H+K]. +ᶟ (m/z 719.6), formed by ETD of [BMP-2+3H+K]+ 4 (m/z 539.8). An asterisk indicates that the ion contains K +. The most abundant ion produced by ETD of quadruply charged [BMP- 2+3H+K]+⁴ (m/z 539.8) is the charge-reduced precursor ion at m/z 719.6 (Figure 5.8), i.e. [BMP-2+3H+K] +ᶟ. This ion was selected for an MS3 experiment via CAD, see Figure 5.9. Most of the fragment ions in Figure 5.9 are doubly charged cn and yn ions and these are different from those present in the ETD and CAD mass spectra of the triply and quadruply charged precursors examined, thus providing complementary sequence insight. 130
All tandem or multi-stage mass spectra presented (Figures 5.4-5.9) corroborate the sequence KIPKASSVPTELSAISTLYL for BMP-2 peptide. [BMP-2+3H]+³ 706.7571 K 84.9708 a₂ 214.9212 [BMP-2+3H+K]+⁴ 539.8031 TELSA-28 474.8460 [BMP-2+2H]+² 1059.6318 100 300 500 700 900 1100 1300 1500 1700 1900 2100 m/z Figure 5.10 ESI-MS spectrum of BMP-2 peptide acquired with the Synapt Q/ToF mass spectrometer. The peptide was dissolved in H2O at 0.01 mg/ml and 10% MeOH + 0.5% formic acid (both v/v %) were added to this solution to enhance the ESI efficiency. The sample preparation protocol was slightly different for the ESI-Q/ToF-MS experiments. BMP-2 was dissolved in a solution of H2O and 10% MeOH plus 0.5 % formic acid (both v/v %) was added to this solution before ESI. No signal was detected on the Synapt Q/ToF instrument without adding formic acid. Figure 5.9 shows the ESI- MS spectrum of BMP-2 peptide acquired with the Synapt Q/ToF mass spectrometer. The most abundant peaks in Figure 5.10 correspond to the same ions as in the ESI-QIT mass 131
spectrum, viz. [BMP-2+nH] +n (n = 2, 3) and [BMP-2+3H+K] +4. Additionally, a few fragment ions are observed, most notably TELSA-28, a2, and K. Figure 5.11 attests that IM-MS removes chemical noise and separates the intact singly, doubly, triply, and quadruply charged peptide from any fragments. The drift times for intact BMP-2 are 7.22 ms for the singly charged ion, 5.26 ms for the doubly charged ion, 2.18 ms for the triply charged ion, and 1.92 ms for the quadruply charged ion. Figures 5.12 shows the drift time distributions of doubly and triply charged BMP-2. It is evident that both ions give rise to single peaks with one drift time, consistent with one sequence and conformation. m/z 3000 [BMP-2]+¹ 2000 [BMP-2]+² [BMP-2]+³ 1000 [BMP-2]+⁴ 1.81 3.61 5.42 7.22 9.03 10.83 12.64 14.44 16.25 drift time (ms) Figure 5.11 2-D IM-MS plot (m/z vs. drift time) of BMP-2 dissolved in H2O at 0.05 mg/ml plus 10% MeOH and 0.5% formic acid (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. 132
(a) [BMP-2+2H] +2 m/z 1059.5720 5.26 0.50 1.50 2.50 3.50 4.50 5.50 drift time (ms) (b) 2.18 [BMP-2+3H] +3 m/z 706.6887 0.50 1.50 2.50 3.50 4.50 5.50 drift time (ms) Figure 5.12 IM-MS chromatograms (drift time distributions) for (a) [BMP-2+2H] +2 (m/z 1059.5720) and (b) [BMP-2+3H] +3 (m/z 706.6887). Figure 5.13 shows the ESI-MS 2 spectrum of triply protonated BMP-2 peptide, viz. [BMP-2+3H] + ³ (m/z 706.76), acquired with the Synapt Q/ToF mass spectrometer. Most fragment ions are singly or doubly charged N-terminal bn sequence ions, as also observed in the CAD spectra measured with the QIT (Figures 5.4 and 5.6) and upon MALDI-MS 2 (Figure 5.2); as has been mentioned, this fragmentation behavior results from the presence of highly basic amino acid residues at and near the N-terminus. 133
Y 136.0815 L 295.1615 PASSVP or PKASSV 86.1034 995.5472 1097.5477 1168.6046 1138.6357 904.0303 KASSVPTEL 1010.5390 1226.7454 ASSVPTELSAIST-H₂O 1251.7473 IPKASSVPTELSA or 1281.7112 PKASSVPTELSAI 1410.7692 1434.770 x₁₃+h 1582.8364 913.0212 IPKASSVPTELSAIST or PKASSVPTELSAISTL PKASSVPTELS PKASSVPTELSA IPKASSVPTE or IPKASSVPTE 838.4581 847.4817 y₂ b₂ 242.1872 PTE 328.1403 a₉ 441.2243 b₁₁ 570.3195 b₁₂ 626.3635 b₁₃-h₂o 660.8760 b₁₄ 705.3888 b₁₅ 762.4427 b₈ 811.5095 100 200 300 400 500 600 700 m/z b₁₇-h₂o b₁₈-h₂o b₁₁ b₁₉ b₁₂ b₁₄ 850 950 1050 1150 1250 1350 1450 1550 1650 1750 m/z Figure 5.13 ESI-MS 2 spectrum of [BMP-2+3H] + ³ (m/z 706.76), acquired with the Synapt Q/ToF mass spectrometer. Singly charged fragments are shown in red color and doubly charged fragments in green color. 134
1373.853 1482.692 1498.717 1412.654 1428.668 5.4 Characterization of HA Peptide This chapter describes the procedure utilized for the analysis of HA peptide. HA is a hydrophilic peptide with fifteen amino acid residues, including four glycine units, three proline and three serine units, two units of valine, and one unit of lysine, methionine, and arginine. The putative sequence of this peptide was GGGSVSVGMKPSPRP. 164 The calculated monoisotopic mass of singly protonated HA is 1412.7278 Da. [HA+O+H]+ [HA+H]+ [y₁₄+h₂o]+ [y₈]+ 867.240 600 700 800 900 1000 1100 1200 1300 1400 m/z Figure 5.14 MALDI-MS spectrum of HA peptide dissolved in H2O at 20 mg/ml. CHCA (20 mg/ml) served as matrix and the sandwich method was used. 135
5.4.1 Characterization of HA by MALDI-ToF/ToF-MS Figure 5.14 shows the MALDI-MS spectrum of HA peptide. The most abundant peaks arise from the protonated ion at m/z 1412.654, [HA+H]+, and an ion at m/z 1428.668 Da, which is 16 Da heavier than the protonated ion and consistent with the composition [HA+O+H] +. Tandem mass spectrometry helps to identify the peptide sequence for both ions. The MALDI-MS 2 spectrum of protonated HA, [HA+H]+ (m/z 1412.654), is shown in Figure 5.15. A contiguous yn series (n=2-11) is observed, as well as selected bn fragments (b8, b14, b14+h2o), all of which are generated by amide bond cleavages. The predominance of the C-terminal yn series is due to the presence of highly basic Arg and Pro residues in the penultimate and terminal positions at the C-terminus. Peptide bond cleavages N-terminal to Pro lead to the most abundant fragments (y3, y5, and b14+h2o), as also observed for BMP-2. The fragmentation pattern in the MALDI- MS 2 spectrum conclusively confirms that the HA peptide has the sequence GGGSVSVGMKPSPRP. The MALDI-MS 2 spectrum of [HA+O+H]+ (m/z 1428.668 ) is shown in Figure 5.16. The dominant fragment at m/z 1365 is formed by loss of a 64-Da moiety, which most likely involves the elimination of CH3SOH from an oxidized methionine side chain (residue no. 7 from the C-terminus). All other fragments are reconciled by consecutive amide bond cleavages in [HA+H-CH3SOH] +, as shown in the scheme on top of the spectrum in Figure 5.16. This supposition is supported by the fact that yn fragments from n=3 until n=6 have the same mass as the corresponding fragments form [HA+H]+ (Figure 5.15), while the larger yn fragments from n=7 until n=11, as well as a14 and b14, have 48- Da smaller masses than the same fragments from [HA+H] + because they are missing part 136
157.007 187.015 272.077 456 601.234 639.281 707.339 313.081 382.112 812.386 869.422 968.500 1055.601 1154.730 1223.781 1269.766 SP-28 VS 212.051 237.043 84.207 KPS KPSP-28 439.149 510.181 536.215 1297.788 PR-NH₃ y₄-nh₃ y₅-nh₃ 664.310 681.346 y₁₂-h₂o 1241.823 1347.763 352.120 y₆-nh₃ y₃-nh₃ 369.137 129.009 1412.654 of the methionine side chain. Note that the 48-Da loss becomes a 64-Da loss after HA oxidation at the Met residue. Since no oxidized peptide was detected by ESI (vide infra), the oxidation must have happened during MALDI. The MALDI matrix used has indeed been found to cause (photo) oxidation at the methionine residue. 178 y₃ y₂ y₅ y₄ y₁₂ y₁₁ y₁₀ y₉ y₈ y₇ y₆ b₁₄+h₂o 1315.794 K HA+H y₃ y₅ 553.239 y₆ y₁₂ b₁₄ y₂ y₄ b₈ x₆ y₇ y₈ y₉ y₁₀ y₁₁ a₁₄ 200 400 600 800 1000 1200 1400 m/z Figure 5.15 MALDI-MS 2 spectrum of protonated HA peptide, [HA+H]+ (m/z 1412.6), acquired on the MALDI-ToF/ToF mass spectrometer. The sequence of the peptide is shown on top of the spectrum, together with the bond cleavages giving rise to the yn series and to b14. 137
157.002 184.989 212.034 237.008 258.986 326.065 608.217 464.107 736.313 764.350 y₇-ch₃soh 792.337 822.384 920.499 942.475 1080.687 1106.728 1221.896 69.940 128.998 PS-28 GGS-H₂O 352.089 396.095 877.416 989.516 1039.514 665.289 707.284 1176.807 VG-28 PR-NH₃ y₃-nh₃ 439.126 510.167 y₈-ch₃soh y₉-ch₃soh y₁₀-ch₃soh y₁₁-ch₃soh a₁₄-ch₃soh 1249.800 b₁₄-ch₃soh 369.111 y₄-nh₃ 681.320 y₁₂ y₁₁ y₁₀ y₉ - CH₃SOH y₈ y₇-ch₃soh y₅-ch₃soh y₆- CH₃SOH y₄-ch₃soh y₃-ch₃soh y₂-ch₃soh [HA+H-CH₃SOH]+ 1365.019 y₅-2 551.198 1428.668 y₃ 200 400 600 800 1000 1200 1400 m/z y₆ R z₆ x₆ b₄ 200 400 600 800 1000 1200 m/z Figure 5.16 MALDI-MS 2 spectrum of [HA+O+H]+ (m/z 1428.668) acquired on the MALDI-ToF/ToF mass spectrometer. Scheme on top of the spectrum shows the product formed after elimination of the oxidized Met side substituent and the consecutive bond cleavages leading to sequence-indicative fragments. 138
[HA+3H]+³ 471.6 [HA+2H]+² 706.9 y₁₀+² 528.2 y₅ 553.2 577.7 y₁₁+² y₁₂+² 621.2 y₁₃+² 649.8 y₈ 869.4 [HA+H]+ 1412.6 200 400 600 800 1000 1200 Figure 5.17 ESI-MS spectrum of HA peptide dissolved in H2O at 0.01 mg/ml + 10% MeOH + 0.5% formic acid (both v/v %), acquired with the QIT mass spectrometer. 1400 m/z 5.4.2 Characterization of HA by ESI-QIT-MS and ESI-Q/ToF-MS The best signal intensities with the QIT were achieved using an aqueous solution of the peptide containing 10% MeOH and 0.5% formic acid (Figure 5.17). The HA peptide forms multiply charged ions under ESI conditions, with the most abundant being the triply protonated peptide at m/z 471.6, [HA+3H]+ 3, and the doubly protonated peptide at m/z 706.9,[HA+2H]+ 2. The intensity of the singly protonated peptide at m/z of 1412.6, [HA+H]+, is low compared to those of the triply and doubly charged ions. Figure 5.17 also includes singly and doubly charged yn fragments like y11 +2, y13, y5, and y8. Oxidation of the methionine residue was not observed in ESI-MS, which confirms that in MALDI- MS this reaction was caused during the MALDI process. 139
y₅ 553.3 705.8 y₈ 869.5 b₁₄+² PR 254.0 SPR-28 323.1 KPSP-H₂O y₃ 369.2 392.1 438.2 y₄-h₂o 456.2 y₄ y₅-nh₃ 536.3507 627.3 a₁₄-nh₃+² 649.4 y₆ 681.4 732.4 754.5 GMKPSPR b₉ y₇ 812.5620 VSVGMKPSP 883.5 y₉ 940.6 b₁₁-h₂o 968.6 y₁₀ 1055.7 y₁₁ 1154.8 200 400 600 800 1000 1200 m/z Figure 5.18 ESI-CAD mass spectrum of doubly protonated HA peptide, [HA+2H] +2 at m/z 706.9, acquired on the ESI-QIT mass spectrometer. The CAD spectrum of the doubly protonated peptide, viz. [HA+2H] +² at m/z 706.9, shows a complete yn series from y3 until y11 (Figure 5.18), which was also present in the MALDI-MS 2 spectrum of [HA+H] + (Figure 5.15) and fully agrees with the HA sequence. Two fragments stand out in this spectrum, y5 from amide bond cleavage N- terminal to Pro, and y8 from amide bond cleavage at the SV-GM junction. The same fragments are also present in the ESI-MS spectrum (Figure 5.17), indicating that they are formed through energetically favorable dissociation pathways. The y8 and y5 fragments from HA (Figure 5.17) were also examined by CAD (Figures 5.19 and 5.20, respectively). The resulting spectra confirm the sequence GMKPSPRP for y8 and PSPRP for y5, further corroborating the sequence of the 8 C-terminal residues of HA. 140
y₅ y₄ y₃ y₂ y₇ y₆ y₈ 869.5 y₈-p y₈-nh₃ PR 253.9 x₂+1 298.9 754.4 GMKPSPR 772.4 352.0 y₃ 369.0 x₃+1 396.0 y₄-h₂o 438.1 x₄+1 483.1 y₅-nh₃ 536.2 y₅ 553.3 GMKPSP+NH₃ 615.3 y₆ 681.4 GMKPSPR-NH₃ 737.4 791.4 839.4 x₇+1 852.4 200 300 400 500 600 700 800 m/z Figure 5.19 ESI-CAD spectrum of the y8 ion (m/z 869.5) in the ESI mass spectrum of HA peptide (Figure 5.17), acquired on the ESI-QIT mass spectrometer. The same spectrum is obtained by an ESI-MS 3 experiment on the y8 ion formed by CAD of [HA+2H] +2 (Figure 5.18). The fragments observed support the composition [GMKPSPRP+H] +. The sequence of HA was also studied by ETD. The spectrum obtained from [HA+3H] +3 (m/z 471.6) (Figure 5.21) shows partially contiguous cn (n=5-13) and zn (n=4-12) fragment series as well as a few yn fragments. Doubly and singly charged HA peptide, formed by proton transfer to the ETD reagent anion, and the loss of H2O or NH3 141
166.9 194.9 237.0 272.0 306.1 334.1 466.2 254.0 352.1 505.2 369.2 PR-NH₃ 420.2 y₃-nh₃ 523.3 PSPR-H₂O 456.2 from the intact peptide and from some fragments are also detected. The fragment ions observed are accounted for by the sequence GGGSVSVGMKPSPRP for the HA peptide. It is noteworthy that c10 and z5 ions are not observed, as these fragments would have required N-C α bond cleavage at a Pro residue, which does not occur upon ETD of multiprotonated peptides (vide supra). When ETD is performed on the y10 +2 fragment from HA peptide (m/z 528.1), the same zn series is observed as for the complete peptide (Figure 5.22), affirming that the 10 C-terminal residues of HA have the sequence SVGMKPSPRP. y₅-h₂o 535.3 y₄ y₃ y₂ PSPR 438.2 y₄-h₂o y₄ PR y₂ y₃ PSP 281.8 y₅ 553.3 200 300 400 500 600 m/z Figure 5.20 ESI-MS 3 spectrum of y5 ion (m/z 553.3) from [HA+2H] +2 (Figure 5.18), acquired on the ESI-QIT mass spectrometer. The same spectrum is obtained by CAD of the y5 ion present in the ESI mass spectrum of HA (Figure 5.17). The fragments observed corroborate the composition [PSPRP+H] +. 142
[HA+3H]+³ 471.4 z₂ 255.8 c₅ 375.0 z₄ 440.0 y₉+² 484.6 c₆ 561.2 853.4 869.4 952.4 796.3 1055.5 z₁₂+² c₈ 613.2 665.3 [HA+2H]+² [HA-NH₃]+² z₆ 749.3 706.8 c₉ 613.2 749.3 z₇ 796.3 z₈ 853.3 GSVSVGMKPS-NH₃ b₁₁-nh₃ z₉ 913.4 940.4 952.5 968.5 c₁₁ y₉ 974.5 z₁₀ 749.3 y₁₀ c₁₃ 1158.6 z₁₂ 1225.6 y₁₃-h₂o 1281.8 1338.7 y₁₄-nh₃ y₁₄-1 [HA+H-NH₃]+ [HA+H]+ 1395.0 1412.7 200 400 600 800 1000 1100 m/z Figure 5.21 ESI-ETD mass spectrum of triply protonated HA peptide, [HA+3H] +3 (m/z 471.6), acquired on the ESI-QIT mass spectrometer. y₁₀+² 528.1 z₂+1 256.9 z₄+1 441.0 z₆ 665.3 z₇ z₈ y₈ y₁₀-h₂o 1037.5 z₉ y₁₀ 200 400 600 800 1000 m/z Figure 5.22 ESI-ETD mass spectrum of the doubly protonated y10 ion (m/z 528.1) in the ESI mass spectrum of HA (Figure 5.17), acquired on the ESI-QIT mass spectrometer. 143
[HA+3H]+³ 471.6241 [HA+2H]+² 706.9467 y₁₁+² y₁₀+² 115.0702 363.4560 413.3062 528.3397 577.8751 y₁₂+² 649.9100 621.4059 y₁₃+² [HA+H]+ 1412.8912 100 300 500 700 900 1100 1300 1500 1700 1900 m/z Figure 5.23 ESI-MS spectrum of HA peptide dissolved in H2O at 0.01 mg/ml + 10% (v/v %) MeOH, acquired with the Synapt Q/ToF mass spectrometer. For the experiments on the Synapt Q/ToF instrument, the HA peptide was dissolved in H2O at 0.01 mg/ml and 10% (v/v %) of MeOH was added to this solution. Figure 5. 23 shows the ESI-MS spectrum acquired from this sample. The most abundant peaks correspond to doubly and triply charged ions of HA, having the compositions [HA+2H] +2 (m/z 707.9467) and [HA+3H] +3 (m/z 471.6241), respectively. The intensity of the singly charged ion [HA+H] + (m/z 1412.8912) is low. Most of the other ions in the same spectrum are fragment ions with different charges. These characteristics are very similar with those in the ESI-MS spectrum measured with the QIT mass spectrometer (Figure 5.17). Figure 5.24 shows the 2-D IM-MS plot obtained by separating all ions formed upon ESI according to their mobilities before mass analysis. The IM dimension removes chemical noise and separates the intact singly, doubly, and triply charged 144
peptide from fragments and aggregates. Under the IM conditions used, the drift times of singly, doubly, and triply protonated HA are 1.53, 3.16, and 12.18 ms, respectively. m/z 3000 2000 [(HA)₄]+⁴ [(HA)₂]+² [(HA)₁]+¹ 1000 [(HA)₁]+³ [(HA)₁]+² 1.81 3.61 5.42 7.22 9.03 10.83 12.64 14.44 drift time (ms) Figure 5.24 2-D IM-MS plot (m/z vs. drift time) of HA dissolved in H2O at 0.01 mg/ml plus 10% (v/v %) MeOH, acquired with the Synapt Q/ToF mass spectrometer. It is evident from Figure 5.24 that the ion at m/z 1412.8 contains at least three different components. This is more clearly visible in the 2-D IM-MS plot acquired from mass-selected m/z 1412.8 under the following conditions: trap collision energy at 6 ev, transfer collision energy at 4 ev, and low mass resolution at 11. Figure 5.25 shows this plot together with the corresponding drift time distribution, both of which reveal the presence of three superimposed ions drifting at 12.18, 7.04, and 3.88 ms. The mass spectra extracted from the three peaks in the drift time chromatogram are depicted in Figure 5.26. Their isotope spacings indicate that the ion with a 12.18 ms drift time is singly charged HA peptide, i.e. [HA+H] + ; the ion drifting at 7.04 ms is doubly charged HA peptide dimer with a molecular weight of 2823.8 Da, i.e. [(HA)2+2H] +2 ; and the ion 145
drifting at 3.50 ms is quadruply charged HA peptide tetramer with a molecular weight at 5647 Da, i.e. [(HA)4+4H] +4. The dimer and tetramer could be formed in solution or during the ESI process. Their observation attests a high tendency for aggregation of HA units, whose basic amino acid residues (Pro, Lys, Arg) and large number of Ser residues enable the formation of strongly hydrogen bonded multimers. (a) m/z 1600 3.50 millisecond 12.18 millisecond [(HA)₄]+⁴ [(HA)₂]+² [(HA)₁]+¹ 1400 7.04 millisecond 1.81 3.61 5.42 7.22 9.03 10.83 12.64 14.44 drift time (ms) 16.25 (b) [(HA)₁]+¹ 12.18 [(HA)₂]+² 7.04 [(HA)₄]+⁴ 3.50 3.00 5.00 7.00 9.00 11.00 13.00 1.00 15.00 Drift time (ms) Figure 5.25 (a) 2-D IM-MS plot (m/z vs. drift time) of m/z 1412.8 from HA peptide after mass selection by the Q mass analyzer. (b) 1-D drift time distribution (chromatogram) extracted from this plot. 146
[(HA)₁]+¹ 1412.8912 1413.8868 12.18 millisecond 1414.8967 [(HA)₂]+² 1412.8912 1413.3890 1413.8868 1414.3708 1414.9104 7.04 millisecond [(HA)₄]+⁴ 1412.5457 1412.8082 1413.3475 1413.9283 1413.1678 1413.5551 1414.2465 1414.4817 1414.8828 3.50 millisecond 1415.2424 1413 1414 1415 m/z Figure 5.26 Mass spectra extracted from each of the three peaks in the drift time chromatogram of m/z 1412.8 from HA peptide. Figure 5.27 shows the ESI-MS 2 spectrum of [HA+3H] + ³ (m/z 471.6341) acquired with the Synapt Q/ToF mass spectrometer. A contiguous series of singly charged yn ions (y3-y10) is present in this spectrum along with a few N-terminal (b2, b3) and internal fragments, all of which agree well with the HA peptide sequence. An MS 2 experiment was also run on the mobility separated [HA+2H] +2 ion (m/z 706.9). Figure 5.28 shows the resulting 2-D IM-MS 2 plot (m/z vs. drift time) and Figure 5.29 the mass spectrum extracted from this plot. Again, a contiguous series of singly charged yn ions (y3-y13) dominates, along with a few N-terminal and internal fragments which verify the sequence GGGSVSVGMKPSPRP for the HA peptide. It should be mentioned at this point that all charge states of HA dissociate to yield predominantly C-terminal yn ions (Figures 5.15, 5.18, 5.27, and 5.29). The number of proton charges influences, however, the relative yn abundances. The most complete sequence coverage is obtained with doubly protonated 147
706.9272 812.5515 852.5585 y₈-nh₃ 869.5570 968.6504 172.0998 484.8146 1055.6786 681.4897 115.0702 187.1349 352.2271 369.2710 GMKPS-NH₃ VS 237.1665 255.1733 y₃-nh₃ 72.0993 87.0723 129.1248 PR-NH₃ y₂-nh₃ 456.3028 HA using Q/ToF instrumentation which minimizes mass discrimination effects (cf. Figures 5.18 and 5.29). y₃ y₂ y₁₂ y₁₁ y₁₀ y₉ y₈ y₇ y₆ y₅ y₄ GS 159.1340 y₅ 553.3663 V a₂ K y₄ b₂ y₃ b₃ y₆ y₈ x₆ y₇ y₉ y₁₀ 100 200 300 400 500 600 700 800 900 1000 1200 m/z Figure 5.27 ESI-MS 2 spectrum of [HA+3H] + ³ (m/z 471.6341), acquired with the Synapt Q/ToF mass spectrometer. 148
(a) m/z 1500 y₁₃ y₁₂ y₁₁ 1000 y₉ y₁₀ y₇ y₈ y₆ 500 y₄ y₅ b₄ (b) 1.81 2.71 3.61 drift time (ms) [HA+2H]+² 706.9467 3.16 4.51 5.42 0.50 1.50 2.50 3.50 4.50 5.50 6.50 Drift time (ms) Figure 5.28 (a) 2-D IM-MS 2 plot (m/z vs. drift time) of [HA+2H] + 2 (m/z 706.9) from HA peptide, acquired with the Synapt Q/ToF mass spectrometer. (b) 1-D drift time chromatogram of m/z 706.9. MS 2 was performed by CAD after the IM separation step. 149
664.4490 852.5477 1038.6584 812.5620 1154.7711 369.2639 456.3185 536.3507 y₆-nh₃ y₈-nh₃ y₁₀-nh₃ 187.1349 y₅-nh₃ 681.4802 968.6390 VS GMKPS-NH₃ 1055.6906 72.0993 V VG-28 129.1248 b₄-h₂o 241.1194 y₅ 553.3749 y₈ 869.5679 b₄ 259.1425 y₁₀ 159.1386 VS-28 y₆ y₉ y₃ y₄ y₇ y₁₁ y₁₂ y₁₃ 100 300 500 700 900 1100 1300 1500 m/z Figure 5.29 Mass spectrum extracted from the 2-D IM-MS 2 plot of [HA+2H] + 2 (m/z 706.9) from HA peptide. 5.5 Characterization of BMP-2(HA)2 hybrid material The synthetic steps used in the laboratory of Prof. M.L. Becker to prepare the BMP- 2(HA)2 hybrid material are explained in Scheme 5.1. 98 The synthesis started with the compound X, which has dendridic structure with two monodisperse polyethylene glycol (PEG) branches terminated by a diazide group and a carboxylic acid functionality at the focal point. The carboxylic acid in X was reacted with the BMP-2 peptide to form compound X-BMP-2. The N-terminus of HA peptide was derivatized with an alkyne terminated substituent to obtain compound Y-HA. Subsequently, click chemistry involving azide-alkyne Huisgen cycloaddition was utilized to link the azide groups in compound X-BMP-2 with the alkyne groups in compound Y-HA in the presence of 150
copper (I) acetate; 1, 3-dipolar cycloadditions between the terminal alkynes and azides take place to form BMP-2(HA)₂ hybrid material in which two HA units are attached to the dendron branches via triazole linkages (Scheme 5.1). The calculated monoisotopic mass of protonated BMP-2(HA)2 [C263H444N69O84S2] is 5977.201035 Da. X BMP-2 X-BMP-2 Y-HA BMP-2(HA)₂ Scheme 5.1 Synthetic procedure to BMP-2(HA)2. 5.5.1 Characterization of BMP-2(HA)2 by MALDI-ToF/ToF-MS Figure 5.30 shows the MALDI-MS spectrum of BMP-2(HA)2 hybrid material, measured using an aqueous solution of 10 mg/ml. Sinapinic acid (SA) (20 mg/ml) served as matrix and the sandwich method was used. The two ions in Figure 5.30 are the 151
singly protonated BMP-2(HA)2 ion at m/z 5977.192, i.e. [BMP-2(HA)2+H]+, and the doubly protonated BMP-2(HA)2 ion at m/z 2989.098, i.e.[bmp-2(ha)2+2h]+². Doubly charged ions are not common in MALDI at the molecular size of this hybrid material, unless special structural features permit it. The relatively high intensity of +2 ions is attributed to the presence of highly basic amino acid residues on each of the three peptide branches. [BMP-2-(HA)₂+H]+ 5977.192 [BMP-2-(HA)₂+2H]+² 2989.098 2000 3000 4000 5000 6000 m/z Figure 5.30 MALDI-MS spectrum of BMP-2(HA)2 hybrid material dissolved in H2O at 10 mg/ml. SA (20 mg/ml) served as matrix and the sandwich method was used. The structure of BMP-2(HA)2 is shown on top of the spectrum. 152
5.5.2 Characterization of BMP-2(HA)2 by ESI-Q/ToF-MS The best signal intensities were achieved using an aqueous solution containing 30% MeOH and 0.5% formic acid (Figure 5.31). BMP-2(HA)2 forms multiply charged ions with +3 to +9 proton charges under ESI conditions. The most abundant peaks in Figure 5.31 arise from charge states +6, +7, and +8; without adding formic acid, the most abundant peaks shift to +5, +6, and +7. [BMP-2-(HA)₂+7H]+⁷ 854.7367 [BMP-2-(HA)₂+6H]+⁶ 997.0446 [BMP-2-(HA)₂+8H]+⁸ 748.0417 [BMP-2-(HA)₂+9H]+⁹ 665.0270 [BMP-2-(HA)₂+5H]+⁵ 1196.2414 [BMP-2-(HA)₂+4H]+⁴ 1495.0475 600 800 1000 1200 1400 1600 m/z Figure 5.31 ESI-MS spectrum of BMP-2(HA)2 hybrid material dissolved in H2O at 0.01 mg/ml + 30% of MeOH + 0.5% formic acid (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. IM-MS analysis spreads the ions formed by ESI into two dimensions (m/z and drift time) according to their mobilities in the traveling wave field (Figure 5.32). Each charge state of intact BMP-2(HA)2 (from +4 to +9) is dispersed into a unique 2-D location, defined by a specific m/z ratio and drift time. Table 5.1 lists the drift times 153
(a) m/z 2000 [BMP-2-(HA)₂]+⁴ [BMP-2-(HA)₂]+³ [BMP-2-(HA)₂]+⁵ 1000 [BMP-2-(HA)₂]+⁸ [BMP-2-(HA)₂]+⁶ [BMP-2-(HA)₂]+⁷ [BMP-2-(HA)₂]+⁹ 1.28 2.57 3.85 5.13 6.41 7.70 8.98 drift time (ms) (b) 3.08 [BMP-2-(HA)₂+4H]+⁴ m/z 1495.0475 4.14 [BMP-2-(HA)₂+5H]+⁵ m/z 1196.2414 4.43 [BMP-2-(HA)₂+6H]+⁶ m/z 997.0446 3.43 [BMP-2-(HA)₂+7H]+⁷ m/z 854.7367 2.85 [BMP-2-(HA)₂+8H]+⁸ m/z 748.0417 2.34 [BMP-2-(HA)₂+9H]+⁹ m/z 665.0270 1.40 1.80 2.20 2.60 3.00 3.40 3.80 4.20 4.60 5.00 drift time (ms) Figure 5.32 (a) 2-D IM-MS plot (m/z vs. drift time) of BMP-2(HA)2 hybrid material dissolved in H2O at 0.01 mg/ml + 30 % MeOH + 0.5% formic acid (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. (b) Drift time chromatograms of intact BMP-2(HA)2 charge states. 154
measured if IM separation is performed at a traveling wave height of 8 V and traveling wave velocity of 250 m/s. Figure 5.32 and Table 5.1 reveal the expected trend for charge states +9 to +6: as the charge decreases, the drift time increases due to decreasing drifting velocity. Further charge decreases, however, to +5 and +4, cause drift time decreases instead. This reversal diagnoses a conformational charge, brought upon by folding of BMP-2-(HA)2 into a more compact structure, as charge repulsion forces are removed. Table 5.1 Drift times of BMP-2(HA)2 ions for IM-MS analysis at traveling wave height and traveling wave velocity of 8V and 300 m/s, respectively. m/z Charge Drift Time 1495.0475 +4 (ms) 3.08 1196.2414 +5 4.14 997.0446 +6 4.43 854.7367 +7 3.43 748.0417 +8 2.85 665.0270 +9 2.34 5.6 Collision cross-sections and molecular modeling The conformation of the dendritic hybrid material was investigated further by determining the collision cross-sections of the different charge states observed by ESI. For this, the drift time scale was calibrated with polyalanine oligomers as standards. Figure 5.33 shows the calibration curve obtained by plotting the corrected drift times of 155
Normalised CCS, Ω (Ų) singly and doubly protonated polyalanine oligomers measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8 V, against their corrected published cross-sections. The actual values used for this plot are summarized in Table 5.2. With the help of this curve (y = 575.62 x 0.4788 where y is the normalized CCS and x is the corrected drift time), the known collision cross-sections of several charge states of ubiquitin and cytochrome C were determined first to test the validity of this calibration. Table 5.3 shows the drift times measured for the ubiquitin and cytochrome C ions, the CCSs derived from these times based on the calibration curve of Figure 5.33, and the corresponding published CCS data. The differences between published CCS and CCS determined in this study are between 3 and 7 %, with seven out of the eight pairs of CCSs lying within 5%. polyalanine 2000 1800 1600 1400 1200 1000 800 600 400 200 0 y = 575.62x 0.4788 R² = 0.9931 0 2 4 6 8 10 12 Corrected drift time, t D (ms) Figure 5.33 Plot of corrected drift times (arrival times) against corrected published cross sections for the +1 and +2 charge states of protonated polyalanine oligomers. Drift times were measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8 V. 156
Table 5.2 Corrected collision cross-section of the polyalanine calibrant ions, [H(Ala)nOH+zH] +2 (z=1-2), deduced from drift times measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8 V. z a n m n-mer (Da) m/z t D b (ms) t D c (ms) Ω (Ų) reduced mass d Ω e (Ų) 1 6 444.15 445.16 1.67 1.6403 146 5.13307 749.428 1 10 728.24 729.25 3.59 3.5519 199 5.19351 1033.509 1 12 870.29 871.30 4.62 4.5784 223 5.20928 1161.670 1 16 1154.70 1155.70 7.05 7.0021 276 5.22940 1443.315 1 20 1438.91 1439.92 9.49 9.4365 337 5.24168 1766.445 1 22 1580.98 1581.99 11.03 10.9739 353 5.24618 1851.901 2 17 1225.42 613.72 1.41 1.3751 265 5.23298 693.370 2 18 1296.43 649.22 1.54 1.5041 276 5.23618 722.594 2 19 1367.45 684.74 1.80 1.7631 287 5.23906 751.806 2 20 1438.76 720.39 1.92 1.8822 297 5.24167 778.388 2 21 1509.51 755.77 2.05 2.0112 308 5.24402 807.579 2 22 1580.54 791.28 2.18 2.1403 317 5.24617 831.517 2 23 1651.55 826.78 2.44 2.3995 327 5.24813 858.069 2 24 1722.59 862.30 2.57 2.5286 337 5.24994 884.614 2 25 1793.61 897.82 2.82 2.7778 348 5.25160 913.779 2 26 1864.64 933.33 3.08 3.0369 358 5.25314 940.312 a For the doubly charged ions, CCS values were taken from reference 139. For the singly charged ions, the CCS values were calculated in this study using the Materials Studio and MOBCAL (projection approximation) programs. The CCS of singly charged ions from reference 139 did not fit into the same line as the CCSs of the doubly charged ions (see Appendix). b measured drift time c t D` = t D - [ 1.41 m z 1000 ] d Reduced mass = m ion m N m ion +m N, where m ion and m N are the masses of the polyalanine ion (see table) and the drift gas molecule (N2, 28 Da), respectively. e Ω`= Ω*( m ion m N m ion +m N /z) 157
Table 5.3 Comparison of collision cross-sections determined using the calibration curve in Figure 5.33 with published collision cross-sections for ubiquitin and equine cytochrome C ions with different numbers of proton charges. z m ion (Da) m/z t D (ms) t D a (ms) Reduced mass b Ω c (Ų) Ω d (Ų) Pubished Ω (Ų) % error e 9 8559.99 952.118 2.95 2.907 5.284 959.394 1634 1519 7 10 8560.09 857.017 2.44 2.399 5.284 875.125 1656 1573 5 11 8560.00 779.190 2.18 2.141 5.284 828.706 1725 1652 4 13 12349.24 950.949 2.82 2.777 5.286 938.608 2308 2193 5 14 12350.01 883.152 2.57 2.528 5.286 897.416 2377 2268 5 15 12349.86 824.332 2.31 2.270 5.286 852.230 2418 2322 4 16 12349.27 772.837 2.18 2.141 5.286 828.735 2508 2438 3 17 12348.67 727.401 2.05 2.012 5.286 804.470 2587 2508 3 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass = m ion m N m ion +m N c Ω` obtained from calibration curve in Figure 5.33 (y =575.62 X 0.4788 ) d Exp Ω =Ω`* (z/ m ion m N m ion +m N e % error = [(Exp Ω- Published Ω)/ Exp Ω]*100 Table 5.4 shows the collision cross-section determined in this study for charge states +4 to +9 of the BMP-2(HA)2 hybrid material. Increasing the charge from +4 to +9 approximately doubles the CCS from 741 to 1463 Ų (+ 97 %). Most of the increase occurs upon adding a proton to the +4 charge state (741 1070 Ų or +44 %). A futher proton (+5 +6) causes a smaller but still substantial CCS increase (1070 1328 Ų or +24 %). More than 6 protons exert only subtle charges in the BMP-2(HA)2 conformation (+2-5 %). These trends strongly suggest that the 3 peptide chains of the hybrid material are tightly folded through a network of inter- and intrachain hydrogen bonds when 4 th proton charges are added. Significant unfolding occurs when a 5 th proton is added and more unfolding is caused when the 6 th proton is added due to charge repulsion. Further 158
proton additions cause very small CCS changes (~ 10% from +6 to +9), indicating that most hydrogen bonding between remote locations has been disrupted with 6 proton charges. Table 5.4 Collision cross-sections of different multiply charged ions of BMP-2(HA)2. z m hybrid (Da) m/z t D (ms) t D (ms) Reduced mass Ω (Ų) Ω (Ų) 4 5976.97 1495.248 3.08 3.02548 5.2800 978.003 741 5 5976.17 1196.241 4.14 4.09123 5.2800 1130.034 1070 6 5976.22 997.045 4.43 4.38548 5.2800 1168.244 1328 7 5976.1 854.737 3.43 3.38878 5.2800 1032.572 1369 8 5976.27 748.042 2.85 2.81144 5.2800 944.241 1431 9 5976.17 665.027 2.34 2.30364 5.2800 858.341 1463 More information on the structure of the BMP-2(HA)2 hybrid material in charge states +4, +5 and +6 was sought by molecular mechanics/dynamics modeling. Proton charges were added to arginine and lysine residues which have the highest proton affinities among the common amino acids found in peptides and proteins (1051 and 996 kj/mol, respectively) 179 as well as to the triazole functionalities. The proton affinities of 1, 4-substituted triazoles, 180 like those present in BMP-2(HA)2, are higher than that of proline (921 kj/mol), tyrosine (926 kj/mol), or methionine (935 kj/mol), 179 which are the other relatively basic amino acid residues in BMP-2(HA)2; hence, the triazoles are also likely protonation sites. BMP-2 peptide contains two lysine units, and each HA peptide contains one arginine and one lysine unit, and each Dendron branch contains one triazole moiety. 159
Table 5.5 Collision cross-sections calculated from energy-minimized structures of average CCSs of BMP-2(HA)2 using the projection approximation (PA), trajectory (TJ), and exact hard sphere scattering (EHSS) methods of the MOBCAL program. z Proton charge position a CCS (Å 2 ) PA EHSS TJ 4 Triazoles (N, N); HA1 (R) ; HA2 (R) 790 965 953 HA1 (K, R); HA2 (K, R) 905 1096 1072 BMP-2 (K, K); HA1 (R); HA2 (R) 923 1115 1055 BMP-2( K); HA1 (R); HA2 (K, R) 933 1126 1067 5 BMP-2 (K, K); HA1 (K, R); HA2 (R) b 882 1058 1072 1264 1056 1145 BMP-2 (K); HA1 (K, R); HA2 (K, R) 920 1112 1067 6 BMP-2 (K, K); Triazole (N); HA1 (K, R); HA2 (R) b 886 1163 1074 1371 1018 1227 BMP-2 (K, K); Triazole (N, N); HA1 (R); HA2 (R) BMP-2 (K, K); HA1 (K, R); HA2 (K, R) 785 908 954 1100 936 1087 a Amino acid residue in BMP-2 peptide and in either of the HA peptide (HA1 and HA2) that is protonated. The natations triazole (N) and triazoles (N, N) indicate that one or both triazole rings in BMP-2(HA)2 are protonated (at an N atom). b There tautomers fold into two different conformers with distinct compactness and CCS (see text). Energy minimization for different tautomers of [BMP-2(HA)2] +4, [BMP- 2(HA)2] +5, and [BMP-2(HA)2] +6 was performed with the Anneal and Geometry Optimization tasks in the Forcite module of the Materials Studio version 7.0 program (Accelrys Software, Inc.). Fitly candidate structures were considered for each tautomer. The number of annealing cycles was 50 for each initially energy-minimized structure. The initial and midcycle temperatures were 50 and 1400 K, respectively; 20 heating ramps per cycle, 1000 dynamics steps per ramp, and one dynamics step per femtosecond 160
were used. The collision cross-sections of the optimized structures were calculated by the projection approximation, exact hard sphere scattering, and trajectory methods available in the MOBCAL algorithm. Depending on the CCS spread among the 50 candidate structures per tautomer, either all 50 CCS values were averaged (if they were grouped together), or the CCSs were divided into families of closely grouped values and each family was averaged individually. The results are summarized in Table 5.5. The hybrid material BMP-2(HA)2 with 4-6 proton charges has 865-867 atoms. At this size, the trajectory method is believed to provide the most reliable CCS predictions. On the other hand, the PA method usually underestimates CCSs of larger ions, especially if they have nonspherical shapes, while the EHSS method tends to overestimate CCSs of systems with < 1000 atoms. The guidelines presuppose that the structure of the ios is simulated accurately; however, as shown in chapter 4, molecular mechanics/dynamics calculations may underestimate the hydrogen bonding network in macromolecular ions that can develop multiple hydrogen bonds, such as multi-protonated BMP-2(HA)2, thus leading to overestimated CCS values by the TJ kmethod and to more accurate predictions by the PA method (due to canceling of errors woth opposite effects). To test whether PA provides more usable CCS results, the structure of bovine insulin with +3 proton charges (774 atoms) was optimized using the Materials Studio software (as described above) and the CCS of the resulting structure was calculated (protons were added to the Arg and the two His residues). The PA, TJ, and EHSS methods of the MOBICAL program yielded 791,950 and 964 Å 2, respectively. The PA value is in very good agreement with the reported value (760 Å 2 ), 181 confirming that PA can provide reliable CCS predictions for 161
Collision Cross-section (Ų) species of rather globular shape that can form extensive hydrogen bonding networks (like insulin and dendritic BMP-2(HA)2. Four different tautomers of BMP-2(HA)2 with +4 charges were considered (Table 5.5). The one protonated at the triazole linkers and the arginine residues of the HA branches has a CCS of 790 Š2 which agrees reasonably (within 6 %) with the experimentally deduced value (Table 5.4). The corresponding energy-minimized structure (Figure 5.34) is globular and quite compact with dense inter- and intra-chain hydrogen bonding interactions. (See Appendix for the structures of the other tautomers.) 1400 1200 1000 800 790 Ų 600 400 200 0 0 50 100 150 200 250 300 350 Relative Potential Energy (kcal/mol) Figure 5.34 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with +4 proton charges at the following sits: Triazoles (N, N); HA1 (R); HA2 (R). The average relative energy and CCS (PA) of all 50 candidate structures are 29 kcal/mol and 790 Ų, respectively. A representative structure is inserted inside the plot. 162
The most stable conformers of BMP-2(HA)2 with +5 charges are protonated on the arginine and lysine residues of the BMP-2 and HA hranches (Table 5.5). For the tautomers with two proton charges on BMP-2, the simulations predict two populations of conformers, differing in compactness (Figure 5.35). The CCS of the less compact structure, 1058 Š2, is strikingly similar with the experimentally determined CCS for this charge state (1070 Š2 ), strongly suggesting that this conformation is probed in the IM- MS experiment. (See Appendix for the structures of another +5 tautomer.) 1400 1200 1000 1058 Ų Collision cross-section (Ų) 800 600 400 887 Ų 200 0 0 50 100 150 200 250 300 350 Relative Potential Energy (kcal/mol) Figure 5.35 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with +5 proton charges at the following sits: BMP-2 (K, K); HA1 (K, R); HA2 (R). The optimized structures can be divided into two conformational families (circles and squares) with average relative energy and CCSs (PA) of 44 kcal/mol and 887 Ų (circles) and 116 kcal/mol and 1058 Ų (squares), respectively. Representative structures for each population are inserted inside the plot. Adding a sixth charge to BMP-2(HA)2 increased significantly its experimentally derived CCS (Table 5.4). Only one tautomer reconciles this trend, viz. the one obtained 163
by adding a proton to one trizole linker of the +5 ion with two proton charges on BMP-2. As with the +5 ion, the +6 ion generated from it by proton addition to a triazole exists in two populations (Figure 5.36), with the less compact one having a CCS of 1163 Š2 (Table 5.5) which is fairly similar with the experimentally determined CCS of 1328 Š2. (See Appendix for the structures of the other +6 tautomers.) 1400 1200 1000 Collision Cross-section (Ų) 800 600 400 868 Ų 1163 Ų 200 0 0 50 100 150 200 250 Relative Potential Energy (kcal/mol) Figure 5.36 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with +6 proton charges at the following sits: BMP-2 (K, K); Triazole (N); HA1 (K, R); HA2 (R). The optimized structures can be divided into two conformational families (circles and squares) with average relative energy and CCSs (PA) of 61 kcal/mol and 868 Ų (circles) and 144 kcal/mol and 1163 Ų (squares), respectively. Representative structures for each population are inserted inside the plot. Theory also predicts the existence of compact tautomers with +5 and +6 charges that are not observed experimentally. It is possible that the protonation sites of these tautomers are not easily accessible in solution sue to the folded structure of BMP-2(HA)2. 164
5.7 Conclusions The results presented in this chapter show that the sequence of BMP-2 and HA peptides is readily determined by using MALDI-MS or ESI-MS 2. The polymer peptide hybrid copolymer BMP-2(HA)2 is characterized by top-down MS, involving tandem MS, (MS n ), and ion mobility mass spectrometry (IM MS). The IM-MS experiments, not only confirm the isomeric purity of BMP-2(HA)2 but also provide clues of its likely protonation sites and overall conformation. Assuming that the lowest charge state (+4) reflects the structure of native hybrid copolymer, this material is folded in aqueous solution through multiple hydrogen bonding interactions. The conformational charges observed by IM-MS from charge states +4 to +6 further indicate that BMP-2(HA)2 should gradually unfold with increasing solution acidity. This intrinsic information might be valuable in the optimization of such hybrid materials for bone regeneration and growth. 165
CHAPTER VI ANALYSIS OF ALKYL POLYGLYCOSIDE (APG) SURFACTANTS USING POLARITY VS. SHAPE SENSITIVE MULTIDIMENSIONAL MASS SPECTROMETRY 6.1 Background A surfactant or surface active agent is active at the surface. Surfactants have the ability to migrate to the surface or the interface between any two immiscible phases and they are categorized by this property. Surfactants reduce the free energy and surface tension of interfaces. 182 A surfactant is an amphiphilic material with polar and non-polar parts. 183 The polar (hydrophilic) part is the head group and the non-polar (hydrophobic) part is the tail group. 184,185 Generally, surfactants are composed of one head and one tail 182 except bolaform and Gemini surfactants which have more than one head or more than one tail. 186-188 Surfactant can have a variety of properties like cleaning, wetting, foaming, emulsifying, dispersing, etc.; 189,190 as a result, they are utilized in many manufactured products like processed foods, pharmaceuticals, personal care and laundry products, 166
petroleum, paints, etc. 191 When they are dissolved in aqueous solution, surfactants have different charges on the hydrophilic head, and based on these charges they are classified into four groups, viz. cationic, anionic, zwitterionic, and nonionic. 182,192 Alkyl polyglycosides (APGs) are a kind of nonionic surfactant which does not produce ions in aqueous solution. APGs contain a sugar head and a hydrophobic hydrocarbon tail. APGs can have more than one glucose unit in the head group and different alkyl chain lengths in their tail. The number of glucose (G) units in the hydrophilic head group is symbolizedbby Gn and the number of carbon atoms (CH2) in the alkyl chain by Cm. 193-195 APGs have low toxicity, fast biodegradability, and they are prepared from renewable resources at low cost. 196-199 Because APGs have good foaming properties, show synergy with other surfactants, and are mild to the skin, they are widely used in cosmetics, household detergents, agricultural and pharmaceutical products, and enhanced oil recovery. 184-186,200-204 APGs are also important used in biological and biomedical research where they are used to dissolve lipid membranes and hydrophobic polypeptides. 134-137 APGs are typically prepared by reacting fatty alcohols with starch or glucose using the Fischer synthesis. 205,206 The Fischer synthesis involves acid-catalyzed acetalization using either direct synthesis or a two-step process. 199,205 The products are very complex because they contain a large number of different chemical compounds. APG products contain mixtures of mono-, di-, and oligoglucosides, mixtures of alkyl chain lengths and mixtures of three types of polysaccharide isomers, including α or β stereoisomers, 1,6- or 1,4-glycosidic bonds and ring isomers with pyranoside or furanoside forms. 199,205,207 167
Different analytical techniques have been used to study APGs. 137 Quantitative analysis of their compesition can be performed by hydrolysis followed by derivatisation with anthrone and calorimetry. 137,208 Fourier transform infrared spectroscopy (FTIR), 1Hnuclear magnetic resonance (NMR), 205,209,207 capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) using pulsed amperometric detection (PAD), 210 thin-layer chromatography (TLC), 211-212 time-of-flight secondary-ion mass spectrometry (ToF-SIMS), 212,213 and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) 207 have been used to study APGs. LC-ESI-MS has been used to separate APGs according to the number of glucose units under normal phase conditions and according to alkyl chain length under reverse phase conditions. 198,204,213,214 High-temperature GC has been used to separate alkyl monoglycosides with isomer resolution and APGs after silylation. 215 Stereoisomers with α or β linkages and ring isomers of APGs has been analyzed by LC-MS using an alkylamide column and isocratic elution. 208 The disadvantages of these analytical techniques are that they are time consuming, require large volumes of organic solvent, and involve many processing steps. In this study, APG surfactant blends have been characterized by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry, tandem mass spectrometry (MS 2 ), and by combining MS and MS 2 with ion mobility (IM) or ultra-performance liquid chromatography (UPLC) separation. The results from both separation techniques (IM vs. UPLC) will be compared to decide which method is best for the precise molecular characterization of APG surfactants. 168
Combination of the two separation methods, via LC-IM-MS experiments, was also examined to evaluate the usefulness of such separation for the identification of minor and trace components in the APG blends. 6.2 Experimental Procedures 6.2.1 MALDI-ToF/ToF-MS MS and MS² experiments were performed in positive mode on a Bruker UltraFlex III MALDI tandem time-of-flight (ToF/ToF) mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a Nd:YAG laser emitting at 355 nm. Dithranol (1,8- dihydroxy-9,10-dihydroanthracen-9) ( 97 %; Aldrich, WI, USA), was used as matrix. A solution of the matrix (20 mg/ml) was prepared in MeOH, and solutions of the APG and NaTFA salt (both 10 mg/ml) were prepared in MeOH (for APG) or in THF (for salt). The matrix and salt solutions were mixed in the ratio 10:1 (v/v %) and this mixture was used for the top and bottom layers of the sample subjected to MALDI. 0.5 1.0 μl of matrix/salt solution were applied first to the MALDI sample target and the droplet was allowed to dry at ambient conditions. After that, 0.5 1.0 μl of APG solution were applied on the top of the matrix/salt spot and allowed to dry at ambient conditions. Finally, 0.5 1.0 μl of matrix/salt solution were applied onto the dried APG spot and allowed to dry at ambient conditions before spectral acquisition. This special preparation, named the sandwich method, led to the formation of [APGs + Na]+ ions. DCTB [Trans-2- (3-(4-tertbutyl phenyl)-2-methyl 2-propenylidene) malononitrile] matrix and AgTFA salt were used for the calibration standard, (PS); [polystyrene, Mn=550], which was analyzed 169
using the dry droplet method instead of the sandwich method. For this, solution of DCTB (20 mg/ml in THF), PS (10 mg/ml in THF), and AgTFA (10 mg/ml in THF) were mixed in the ratio 10:2:1 (v/v/v %), and ~ 0.5 μl of the final mixture were applied onto the MALDI sample target and allowed to dry before the target was inserted into the vacuum system for MALDI. 6.2.2 ESI-QIT-MS MS and MS² experiments were performed on a quadrupole ion trap (QIT) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with an electrospray ionization (ESI) source operated in positive ion mode. The APG sample solutions were injected into the instrument by direct infusion at a flow rate of 3 μl/min. The cone voltage was held at 3.5 kv, the nebulizer gas pressure at 10 psi, and the drying gas flow rate and temperature at 8 L/min and 300 C, respectively. The APG samples were dissolved in H2O at 0.01 mg/ml and 10% (v/v %) of MeOH was added to this solution to improve spray conditions. The APG solutions were mixed with a solution of NaTFA or LiTFA in THF (0.1 mg/ml) in the ratio 100:2 (v/v %) to aid the formation of sodiated or lithiated ions, respectively. 6.2.3 ESI-Q/ToF-MS MS and MS² experiments were performed on a Waters Synapt HDMS quadrupole/time-of-flight (Q/ToF) mass spectrometer (Waters, Beverly, MA, USA), equipped with electrospray ionization (ESI) source. For ESI-MS and IM-MS analysis of 170
the APGs, the samples were dissolved in water at 0.05 mg/ml and 10% of MeOH (v/v) was added to these solutions to improve spray conditions. A solution of NaTFA salt (0.01 mg/ml) was mixed with each APG solution in the ratio 1:100 (v/v). The final mixture was introduced to the ESI source by direct infusion at a flow rate of 10 μl/min. The instrument was operated in positive ion mode with a capillary voltage of 3.15 kv, cone voltage of 35 V, sampling cone voltage of 3.2 V, source temperature of 80 C, and desolvation temperature of 150 ºC. Ion mobility separation was achieved by tuning the wave height and the wave velocity in the triwave ion mobility cell to 8 V and 200 m/s, respectively; the wave height and wave velocity in the trap cell were set at 0.5 V and 300 m/s, respectively; the wave height and wave velocity in the transfer cell were set at 0.2 V and 248 m/s, respectively. The nitrogen gas (drift gas) flow rate in the ion mobility cell was 22 l/h. Tandem mass spectrometry experiments were performed in the transfer cell, located after ion mobility chamber, using argon as collision gas. 6.2.4 Ultra-Performance Liquid Chromatography (UPLC) UPLC separation was performed on a Waters ACQUITY TM UPLC system equipped with a Waters ACQUITY UPLC BEH C18 1.7 μm 1.0x150 mm column with pore size of 130 Å (Part No. 186002350, Serial No. 019431208157). Elution was completed in 3 minutes by using a flow rate of 0.250 ml/min. The sample for UPLC-MS analysis was prepared by dissolving the APGs in MeOH: water (50:50, v/v) at the concentration of 0.1 mg/ml, no salt was added. The APGs were eluted with water + 0.1 % formic acid (mobile phase A) and MeOH: isopropanol (50:50, v/v) + 0.1 % formic 171
343.130 371.228 751.412 505.239 561.318 991.408 1047.470 1125.426 1153.464 477.230 667.303 723.364 829.354 885.417 1209.522 1181.493 1343.538 1505.581 963.377 533.288 1019.438 639.273 801.322 695.334 857.386 acid (mobile phase B) via a gradient program. The mobile phase composition was held at 40% H₂O and 60% MeOH: IPA (v/v) in the first minute and changed to 30% H₂O and 70% MeOH: IPA (v/v) in the next minute. After that, the mobile phase composition was changed from 20% H2O and 80% MeOH: IPA (v/v) to 10% H2O and 90% MeOH: IPA (v/v) in last minute. The UPLC-IM-MS experiments were performed at the same UPLC-MS conditions (same mobile phase, column, time, and flow rate and at the same traveling wave velocity, traveling wave height, and ion mobility gas flow setting used in the IM experiments (vide supra). G₁Na+ $ 399.280 G₃Na+ # G₄Na+ # G₂Na+ #!! G₅Na+ #! @ $ @ $! G₆Na+ @ # @ $ % @ $! # @ $ G₇Na+ # G₈Na+ #! @ $ 400 600 800 1000 1200 1400 m/z Figure 6.1 MALDI-MS spectrum of 818 UP alkyl polyglucoside using the sandwich method and DIT as matrix. The number of glucose units in each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆. 172
G₃Na+ G₁Na+ $ 399.288 G₂Na+ # 533.294 # 695.343 # G₄Na+ 857.396 # 371.256! 477.232 $ $ 561.325 723.374 % % 589.358 751.406 $ 885.429 G₅Na+ # 1019.453 $ 1047.484 % G₆Na+ # 1181.506 $ 1209.535 G₇Na+ # 1343.560 400 600 800 1000 1200 m/z Figure 6.2 MALDI-MS spectrum of 1200 UP alkyl polyglucoside using the sandwich method and DIT as matrix. The number of glucose units in each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆. 6.3 Characterization of APGs by MALDI-ToF/ToF-MS 6.3.1 MALDI-MS of Alkyl Polyglucosides (APGs) More than one matrix was used to analyze the samples by MALDI. DHB and DIT gave the best signal-to-noise ration for the APG compounds. The highest intensities were obtained when DIT was used as matrix. Sample preparation using the sandwich method or the dry droplet method with a mixing ratio of matrix: sample: salt of 10:5:1 gave the same results. The cationizing agents used were NaTFA and LiTFA and both of these salts 173
gave the same results. A simpler spectrum is obtained with Na salt since the samples already contain Na ions originating from the glassware used in their synthesis and storage. G₂Na+! 477.241 G₃Na+! 639.287 # # G₁Na+ $ #371.248 399.288 @ 343.215 @ 505.269 533.299 $ 561.331 @ 667.318 695.348 $ 723.382 % 751.412 G₄Na+! 801.341 @ 829.373 # 857.405 $ 885.435! 963.398 G₅Na+ @ 991.429 # $ 1019.462 1047.494 G₆Na+! 1125.453 1153.485 @ # 1181.517 G₇Na+ # 1343.568 400 600 800 1000 1200 1400 m/z Figure 6.3 MALDI-MS spectrum of 2000 UP alkyl polyglucoside using the sandwich method and DIT as matrix. The number of glucose units in each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆. The MALDI mass spectra of the three APG samples investigated (plantacare 818, plantaren 1200 and plantaren 2000) show a distribution of peak groups that are separated by 162 Da, which is the mass of the glucose repeat unit (Figures 6.1-6.3). The difference between adjacent ions of the same peak group is 28 Da, which is the CH2CH2 repeating unit of the fatty alcohol alkyl chain. In order tofacilitate spectral interpretation, the masses of several APG oligmers were calculated based on the following assumptions: 174
i) Natural fatty alcohols contain an even number of CH2 moieties and these alkyl chains are linear, 216 i.e. (CH2)n where n is an even number from 6 to 20. ii) The glucose unit has a mass of 162 Da. The number of glucose units in the head group was set between 1 and 10, i.e. (G)m where G is the glucose unit (162 Da) and m is the number of glucose units (1-10). iii) The oxygen supplied by the fatty alcohol and the end groups (2H) add together 16+1+1=18 Da (elements of water). Table 6.1 shows the calculated masses for [APG+Na] +1. All samples form [APG+Na] + ions irrespective of whether a sodium salt is added. [APG+Li]+ ions are detected only when lithium salts are added to the APG solutions. Oligomers with varying numbers of glucose (162 Da) and hydrocarbon (28 Da) repeat units and the same end groups (2H, O) are present in all mass spectra. Plantacare 818 is found to contain 1 to 8 glucose (G1-G8) units and C8-C16 alkyl chains (Figure 6.1). Plantaren 1200 gives a narrower distribution, viz. G1-G7 and C12-C14, respectively (Figure 6.2). On other hand, Plantaren 2000 shows G1-G6 and C8-C14 distributions (Figure 6.3). MALDI-MS 2 experiments on select ions gave further evidence on the number of glucose units and alkyl lengths of the APGs studied. 175
Table 6.1 Calculated masses for the [APG+Na]+ ions of APGs mass ([GmCn+Na]+) = n * 162.05275 + m * 14.01565 + 22.9898 (Na+) + 18.01055 (H₂O). G₁ G₂ G₃ G₄ G₅ G₆ G₇ G₈ G₉ G₁₀ (CH₂)₄ 259.1 421.2 583.2 745.3 907.3 1069.4 1231.4 1393.5 1555.5 1717.6 (CH₂)₆ 287.2 449.2 611.3 773.3 935.4 1097.4 1259.5 1421.5 1583.6 1745.6 (CH₂)₈ 315.2 477.2 639.3 801.3 963.4 1125.4 1287.5 1449.6 1611.6 1773.7 (CH₂)₁₀ 343.2 505.3 667.3 829.4 991.4 1153.5 1315.5 1477.6 1639.6 1801.7 (CH₂)₁₂ 371.2 533.3 695.3 857.4 1019.5 1181.5 1343.6 1505.6 1667.7 1829.7 (CH₂)₁₄ 399.3 561.3 723.3 885.4 1047.5 1209.5 1371.6 1533.6 1695.7 1857.8 (CH₂)₁₆ 427.3 589.4 751.4 913.5 1075.5 1237.6 1399.6 1561.7 1723.7 1885.8 (CH₂)₁₈ 455.3 617.4 779.4 941.5 1103.6 1265.6 1427.6 1589.7 1751.8 1913.8 (CH₂)₂₀ 483.4 645.4 807.5 969.5 1131.6 1293.6 1455.7 1617.7 1779.8 1941.8 6.3.2 MALDI-MS 2 of Alkyl Polyglucosides (APGs) Fragments obtained from polysaccharides and glycolconjugates are generally named as shown in Figure 6.4. Fragments containing the non-reducing glucoseterminated side are named A n, B n and C n and fragments containing the alkoxylated reducing side are named Y n, X n and Z n where n is the number of partial or complete glucose repeat units. 217-218 In tandem mass spectrometry (MS 2 ) experiments, the sodiated APG oligomers undergo losses that identify both the saccharide unit as well as the alkyl chain (R), which is eliminated as a dehydrated moiety and ROH alcohol. 176
Y₂ Z₂ ¹ ⁵X₁ Y₁ Z₁ ¹ ⁵X₀ Y₀ Z₀ ⁰ ²A₁ B₁ C₁ B₂ C₂ B₃ C₃ Figure 6.4 Nomenclature of glyco-conjugate fragment ions. Figure 6.5 and Figure 6.6 show the MALDI-MS 2 spectra of G₆C₁₂Na+ (m/z 1181.564) from sample 818 and G₄C₁₂Na+ (m/z 857.445) from sample 1200. All APG ions dissociate by the same pathways, forming B and Y fragments, which are produced by breaking the glycosidic bonds. The Y ions observed contain glucose units attached to the alkyl tail and the B ions contain only glucose units without the tail. The Bn and Yn fragments observed in Figure 6.5 can be rationalized by competitive and consecutive glycosidic bond cleavages, as depicted in Scheme 6.1. Another example of the MALDI- MS 2 fragmentation behavior of APGs is given in Figure 6.6 which shows the spectrum obtained from G₄C₁₂Na+ (m/z 857.445); again, the same types of fragment ions are generated via the the pathway in Scheme 6.1. 177
Y₅ G₅C₁₂Na+ Y₅ Y₄ Y₃ Y₂ Y₁ Y₀ 1019.637 B₁ B₂ B₃ B₄ B₅ B₆ Y₄ G₄C₁₂Na+ 857.467 G₆C₁₂Na+ B₁ G₁Na+ 185.104 Y₁ G₁C₁₂Na+ 389.897 B₃ B₂ G₃Na+ G₂Na+ 509.142 365.078 B₄ G₄Na+ Y₂ 671.195 G₂C₁₂Na+ 533.297 Y₃ G₃C₁₂Na+ 695.352 B₅ G₆Na+ 833.290 B₆ G₆Na+ 995.444 1181.564 200 400 600 800 1000 m/z Figure 6.5 MALDI-MS 2 spectrum of [G₆C₁₂Na]+ ion at m/z 1181.564 from 818 UP sample. Y₃ Y₂ Y₁ Y₀ G₄C₁₂Na+ 857.445 B₁ B₂ B₃ B₄ Y₃ G₃C₁₂Na+ 695.422 B₁ G₁Na+ 185.104 B₂ G₂Na+ 347.174 Y₁ G₁C₁₂Na+ 371.311 B₃ G₃Na+ Y₂ G₂C₁₂Na+ 533.369 B₄ G₄Na+ 671.250 100 200 300 400 500 600 700 800 m/z Figure 6.6 MALDI-MS 2 of 857.445 [G₄ C₁₂Na]+ ion at m/z 857.445 from 1200 UP sample. 178
Y b Y 0 B a B a+b Scheme 6.1 General fragmentation pathways of APGs upon MALDI. The Bb fragment is an internal fragment, but indistinguishable from B-type fragments because the nonreducing end is not substituted. According to the mechanism in Scheme 6.1, breaking any glycosidic bond produces complementary Y and B ions. B ions carry glucose units only and can continue to lose glucose units by cleavage of any glycosidic bond inside their chain. One or more glucose units may be lost in one step. Y ions carry the fatty tail and, hence, are truncated 179
APGs. They can continue to undergo glycosidic bond cleavages that yield either shorter Y fragment ions or shorter B fragment ions. The B fragments produced in the latter step are technically internal fragments, but indistinguishable from shorter terminal B-type fragments. 6.4 Analysis of alkyl polyglucosides (APGs) by ESI-QIT-MS and ESI-Q/ToF-MS 6.4.1 ESI-MS of Alkyl Polyglucosides (APGs) Comparable intensities were achieved using aqueous solutions containing 10% MeOH and NaTFA or LiTFA or no salt. For lithiation, a LiTFA solution (0.1 mg/ml) was mixed with the APG solution in the ratio 2:100 (v: v %). The ESI mass spectra acquired this way for plantacare 818, plantaren 1200, and plantaren 2000 by ESI-QIT-MS (Figures 6.7, 6.8, and 6.9, respectively) are more complex than the corresponding MALDI mass spectra. The ESI mass spectra show abundant [M+Na] + quasi-molecular ions only for the G₁ and G₂ distributions (Figures 6.7, 6.8, and 6.9). The longer polyglycosides (G₃, G₄, G₅, G₆, and G₇) give rise to ions with low intensities and new ions appear in their m/z window. The mass difference between new ions and the nearest APG ions is 4 Da. For example, new ion at m/z 719 is observed 4 m/z units (i.e. 4 Da) below G₃C₁₄Na+ (m/z 723). The new ions are also observed in ESI-Q/ToF-MS spectra. As will be shown, these ions are dimeric complexes. It is noteworthy that sodiated ions dominate the mass spectra in Figures 6.6-6.9, even though a Li + salt was added to the samples. Evidently, APGs have high binding affinities for Na + which is always present when glassware is used for sample preparation or storge. 180
G₃C₁₀ Na+ 675.5 G₃C₁₂ Li+ 731.5 607.3 635.4 647.4 703.5 639.3 G₃C₈ Na+ 695.4 G₁DI Na+ G₁DI Na+ G₁DI Li+ G₁DI Li+ G₃C₁₂ Na+ G₁DI Na+ 663.4 G₁DI Na+ 719.5 G₁DI Na+ 691.4 G₁DI Na+ 747.5 G₁DI Li+ 619.4 G₁DI Li+ 723.4 G₃C₁₄ Na+ # 371.2 G₁! 315.2 G₂ # G₃+G₁ DI @ 343.2 $ 399.2! 477.2 @ 533.3 $ 663.4 691.4 719.4 747.5 % 505.3 561.3 % 427.3 607.3 775.5 857.4 891.3 200 300 400 500 600 700 800 900 1000 m/z G₁DI Li+ 600 620 640 660 680 700 720 740 m/z Figure 6.7 ESI-MS spectrum of 818 UP alkyl polyglucoside dissolved in H2O at 0.01 mg/ml + 10 % MeOH + 2 % LiTFA (both v/v %), acquired by QIT-MS. The number of glucose units in each ion is labeled by Gn, and APG dimers are labeled by Gn DI. The number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆. 181
633.4 663.4 736.5 G₁DI Na+ 681.7 723.4 708.6 G₃C₁₄ Na+ 731.5 G₃C₁₆ Li+ 692.6 695.4 703.5 G₁DI Li+ G₃C₁₂ Na+ G₁DI Li+ 747.4 719.5 G₁ # 371.2 360.4 399.3 G₂ $ # G₃+G₁ DI 533.3 # $ 517.4 561.4 633.4 719.5 747.5 G₄+G₂ DI # 857..4 907.4 G₅+G₃DI # 1019.5 200 400 600 800 1000 m/z G₁DI Na+ G₁DI Na+ 620 640 660 680 700 720 740 m/z Figure 6.8 ESI-MS spectrum of 1200 UP alkyl polyglucoside dissolved in H2O at 0.01 mg/ml + 10 % MeOH + 2 % LiTFA (both v/v %), acquired by QIT-MS. The number of glucose units in each ion is labeled by Gn, and APG dimers are labeled by Gn DI. The number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆. 182
679.5 723.4 615.6 731.5 623.3 685.5 747.4 667.3 G₃C₁₂ Li+ 695.4 703.5 G₃C₁₄ Na+ G₃C₈ Li+ G₁DI Li+ 619.4 G₃C₁₀ Na+ 607.3 635.4 G₁DI Na+ 639.3 G₃C₈ Na+ 675.5 G₃C₁₂ Na+ G₁DI Li+ G₁DI Li+ G₁DI Na+ 647.5 G₁DI Li+ G₁DI Li+ 607.4 533.3 691.4 663.4 719.4 1045.7 891.3 477.2 398.3 343.2 #371.2 315.2! G₁ @ $! G₂ @ # G₃+G₁ DI G₄+G₂ DI G₅+G₃DI 200 400 600 800 1000 1200 m/z G₁DI Na+ 663.4 G₁DI Na+ 691.4 G₁DI Na+ 719.5 651.3 G₃C₁₀ Li+ G₁DI Na+ 620 640 660 680 700 720 740 m/z Figure 6.9 ESI-MS spectrum of 2000 UP alkyl polyglucoside dissolved in H2O at 0.01 mg/ml + 10 % MeOH + 2 % LiTFA (both v/v %), acquired by QIT-MS. The number of glucose units in each ion is labeled by Gn, and APG dimers are labeled by Gn DI. The number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)10, # = (CH₂)₁₂, $ = (CH₂)₁₄, and % = (CH₂)₁₆. 183
In the ESI-Q/ToF experiments, a solution of NaTFA salt (0.01 mg/ml) was mixed with the APG solution (0.05 mg/ml in H2O + 10% MeOH) in the ratio 1:100 (v: v %). The ESI mass spectra of plantacare 818, plantaren 1200, and plantaren 2000 measured this way (Figures 6.10, 6.11, and 6.12, respectively) show the same types of ions as the ESI-QIT mass spectra, although the high mass ions (mostly dimers) are more intense with the ESI-Q/ToF instrument because the solutions of the APGs samples are more concentrated. As with ESI-QIT-MS, [M+Na]+ quasi-molecular ions dominate only for the G₁ and G₂ distrubitions (Figures 6.10, 6.11 and 6.12). The [M+Na]+ ions of the longer polyglycosides (G₃, G₄, G₅, G₆, and G₇) have low intensities and overlap with the new ions. In section 6.3.2.3, ESI-MS 2 of alkyl polyglucosides (APGs), it will be shown that the new ions are sodium bound APG dimers. ESI-IM-MS data (vide infra) further reveal that the dimer intensities increase linearly with the APG concentration in the solutions being electrosprayed, strongly suggesting that the dimers are already formed in solution. ESI-Q/ToF-MS of 818 UP indicates the presence of G1-G8 chains carrying C8-C16 alkyl substituents, as well as dimer ions such as G₁-DI, G₁G₂-DI, G₂-DI, G₂G₃-DI, G₁G₄-DI etc. (Figure 6.10). In plantaren 1200, APGs with 1 to 7 glucose (G1-G7) units and C12-C14 alkyl chains are detected, as well as dimer ions (Figure 6.11). Finally, plantaren 2000 shows G1-G6 blocks that are attached to C8-C14 chains, plus dimer ions (Figure 6.12). 184
619.4 769.3372 797.3714 667.2347 607.2905 635.3186 G₁DI Na+ G₃C₁₀ Na+ G₁DI Na+ 639.2004 150 300 450 600 750 900 1050 1200 1350 1500 1650 m/z G₃C₈ Na+ G₁DI Na+ 663.3350 G₁DI Na+ 691.3657 G₁DI Na+ 719.3940 G₁DI Na+ 747.4260 G₃C₁₂ Na+ 695.2593 G₃C₁₄ Na+ 723.2965 G₁DI Na+ 775.5431 G₄C₈ Na+ 801.2505 610 630 650 670 690 710 730 750 770 790 m/z Figure 6.10 ESI-MS spectrum of 818 UP alkyl polyglucoside dissolved in H2O at 0.05 mg/ml + 10 % MeOH + 1 % NaTFA (both v/v %), acquired with Q/ToF-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI. 185
723.3459 G₃C₁₄ Na+ 200 400 600 800 1000 1200 1400 1600 m/z G₁DI Na+ 719.4433 G₁DI Na+ 747.4763 G₃C₁₂ Na+ 695.3079 G₁DI Na+ 775.5226 G₁DI Na+ G₁DI Na+ 663.4108 691.4333 G₄C₈ Na+ 803.5638 610 630 650 670 690 710 730 750 770 790 m/z Figure 6.11 ESI-MS spectrum of 1200 UP alkyl polyglucoside dissolved in H2O at 0.05 mg/ml + 10 % MeOH + 1 % NaTFA (both v/v %), acquired with Q/ToF-MS. The number of glucose units for each ion is labeled by Gn, and APG dimers are labeled by Gn DI. 186
619.4 769.4189 797.4544 775.5431 639.2656 150 300 450 600 750 900 1050 1200 1350 1500 1650 m/z G₁DI Na+ 663.4014 G₁DI Na+ G₁DI Na+ 635.3837 G₃C₈ Na+ G₁DI Na+ 691.4333 G₁DI Na+ 719.4728 607.3449 G₃C₁₀ Na+ 667.3012 G₃C₁₂ Na+ 695.3370 G₁DI Na+ 747.5166 G₁DI Na+ G₄C₈ Na+ 801.3443 G₃C₁₄ Na+ 723.3658 610 630 650 670 690 710 730 750 770 790 m/z Figure 6.12 ESI-MS spectrum of 2000 UP alkyl polyglucoside dissolved in H2O at 0.05 mg/ml + 10 % MeOH + 1 % NaTFA (both v/v %), acquired with Q/ToF-MS. The number of glucose units for each ion is labeled by Gn, and APG dimer are labeled by Gn DI. 187
133.2 139.0 109.0 150.9 187.0 193.1 249.2 277.2 319.2 235.2 265.2 295.3 337.2 -H₂O -H₂O -H₂O -H 2 O-CO G₁C₁₂ -2H₂O G₁C₁₂ -H₂O 6.4.2. ESI-MS 2 of alkyl polyglucosides (APGs) Figure 6.13 shows the ESI-MS 2 spectrum of G₁C₁₂Li+ (m/z 355.2) from the 1200 sample, acquired on the ESI-QIT mass spectrometer. The parent ion (at m/z 355.2) contains one glucose unit and a C12 fatty chain. From Figure 6.13, it is evident that both cleavages at the glycosidic bond as well as cross-ring cleavages occur, leading to fragments that corroborate the presence of a C12 alkyl chain (Y0) and its attachment at glycosidic OH ( A 0,2 1, X 0,2 0, X 1,4 0, and X 2,4 0 ). y₀ G₁C₁₂Li + B₁ C₁ 168.9 355.2-2H₂O 126.9 100 150 200 250 300 m/z Figure 6.13 ESI-MS 2 spectrum of G₁C₁₂Li+ (m/z 355.2) from the 1200 sample acquired on the ESI-QIT mass spectrometer. 188
Figure 6.14 shows the ESI-MS 2 spectrum of G₂C₈Li+ (m/z 461.2) from the 2000 sample, acquired on the ESI-QIT mass spectrometer. The fragments from glycosidic bond cleavages (B1, C1, Y1, and B2) confirm the G2 and C8 composition, while the fragments from cross-ring cleavages (A and X series) confirm the 1, 4 connectivity of the two glucose units. G₂C₈ Li+ 461.2 Y₁ B₁ C 1 B₂ 299.2 331.1 179.2 126.9 168.9 187.0 209.1 229.0 259.0 281.1 289.1 341.2 371.2 387.3 401.2 443.3 100 200 300 400 m/z Figure 6.14 ESI-MS 2 spectrum of G₂C₈Na+ (m/z 477.2) from the 2000 sample acquired on the ESI-QIT mass spectrometer. Figure 6.15 shows the ESI-MS 2 spectrum of G₃C₈Na+ (m/z 639.3) from the 818 sample, acquired on the ESI-QIT mass spectrometer. Again, the fragmentation pattern observed fully supports a triglycoside structure with a C8 alkyl chain and 1, 4 connectivity in the polysaccharide block. 189
[G 3 C 8 ]Na+ 639.3 Intens. +MS2(639.3), 0.2-0.8min #(2-11) 1500 1000 477.2 500 184.8 244.9 304.9 347.0 364.9 467.1 x15 00 315.1 387.2 407.0 437.1 509.1 519.1 527.2 549.2 579.1 621.3 200 250 300 350 400 450 500 550 600 650 m/z 100 200 300 400 500 600 m/z Figure 6.15 ESI-MS 2 spectrum of G₃C₈Na+ (m/z 639.3) from the 818 sample acquired on the ESI-QIT mass spectrometer. The MS 2 spectra of the new ions reveal that they are metal-cationized dimers, [2M+Na]+ or [2M+Li]+, containing two APG molecules. This is exemplified in Figure 6.16 by the ESI-MS 2 spectrum of the G₁ DI Li+ ion (m/z 703.5) from the 1200 sample, acquired on the ESI-QIT mass spectrometer. The dimer decomposes into its monomer components, which include G1C8, G1C10, G1C12, G1C14 and G1C16. The fragments observed clearly show that three different isomeric dimers overlap at m/z 703.5, viz. the homodimer [G1C12]2 Li + and the heterodimers [G1C8+G1C16] Li +. The same composition is identified in the MS 2 spectrum of the corresponding sodiated dimer G1 DI Na + at 719.4 (Figure 6.10). On the other hand, the G1 DI Na + dimer at m/z 663.4 (Figure 6.10) shows 190
299.2 372.2 411.3 G₁ C₈Li+ 383.3 G₁ C₁₀Li+ G₁ C₁₆Li+ G₁ C₁₄Li+ only one fragment in its MS 2 spectrum, indicating a single composition, viz. [G1C10]2Na + (see also IM-MS section below). Hence, MS 2 provides a convenient means to ascertain in the varieus dimers formed in the APG solutions. Dimers are observed at all sample concentrations examined, which ranged from 0.001 to 0.1 mg/ml APG. Within this concentration regione, dimer ion intensities increase linearly with the APG solution concentration, as attested by ESI-IM-MS (vide infra). This trend strongly suggests that hydrogen bonded dimers are already present in solution, consistent with the micelle-forming properties of APGs. G₁ C₁₂Li+ 355.2 (G₁ C₁₂)₂Li+ (G₁ C₁₀ +G₁ C₁₄) Li+ (G₁ C₈ +G₁ C₁₆) Li+ 703.5 100 200 300 400 500 600 700 m/z Figure 6.16 ESI-MS 2 spectrum of the G₁ DI Li+ (m/z 703.5) from the 1200 UP acquired on the ESI-QIT mass spectrometer. 191
m/z [G n C m ] + [G n C m ] x (APG + multimers) 3000 2000 [G n C m ] 2 +1 (APG monomers) 1000 [G n C m ] +1 (APG dimers) 1.81 3.61 5.42 7.22 9.03 10.83 12.64 drift time (ms) Figure 6.17 2-D IM-MS plot (m/z vs. drift time) of the 818 UP dissolved in H2O + 10 % MeOH at 0.05 mg/ml + 1 % NaTFA (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. 6.5 ESI-IM-MS and ESI-IM-MS 2 of Alkyl Polyglucosides (APGs) Ion mobility (IM) spectrometry was used to separate the APGs from their dimers (and higher order aggregates) species have different shapes (architectures). Figure 6.17 shows the 2-D IM-MS plot (m/z vs. drift time) of 818 UP, obtained by sending all ions formed in the ESI source through the Triwave IM region, where they are separated by charge, size, and shape, and subsequent ToF mass analysis of mobility-separated ions. APG ions and dimer carrying one charge are separated into different mobility regions because the dimers have less compact structures (larger collision cross-sections) than monomeric. APGs with comparable mass, this drifting more slowly through the IM 192
885.3 991.5 829.4 963.4 1047.5 1019.5 1125.5 1153.5 1181.6 1209.6 589.3 723.4 857.3 427.3 667.2 801.2 505.2 561.2 695.4 639.2 343.2 399.2 477.2 533.2 315.2 371.2 region. Figure 6.18 shows the mass spectrum extracted from the IM region of singly charged APG in the IM-MS plot of 818 UP alkyl polyglucoside and Figure 6.19 shows the mass spectrum extracted from the IM region of singly charged X-mers (dimers) from this sample. The mass spectrum of mobility-separated monomeric APG (Figure 6.17) readily discloses both the glycoside and alkyl chain distributions of plantacare 818. It is quite similar with the corresponding MALDI-MS spectrum (Figure 6.1). The mass spectrum of the mobility separated dimers (Figure 6.19) further shows that monoglycosides (G1) have the highest tendency to aggregate into dimers. G₁Na+! # G₂Na+ @ $! # G₃Na+! @ $ # G₄Na+ % % @ $! @ # $ G₅Na+ #! $ @! G₆Na+ # @ $ 200 300 400 500 600 700 800 900 1000 1100 m/z Figure 6.18 Mass spectrum extracted from the [APG]+ 1 mobility region in the ESI-IM- MS plot of 818 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)12, # = (CH₂)₁₄, $ = (CH₂)₁₆, and % = (CH₂)₁₈. 193
G₁DI 775.5 635.3 747.4 607.3 691.4 663.3 719.4 (G₁+G₂)DI G₂ DI 587.5 803.5 831 853.4 881.5 909.5 937 987.4 1015.5 1043.5 1071.5 (G₁+G₄ and G₂+G₃)DI 550 650 750 850 950 1050 m/z Figure 6.19 Mass spectrum extracted from the [X-mer] +1 mobility region in the ESI-IM- MS plot of 818 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn. Figure 6.20 shows the 2-D IM-MS plot (m/z vs. drift time) of the 1200 UP APG sample under ESI conditions. Again, APG monomer dimer ions with one charge (Na + ) are separated efficiently in the IM dimension. Figures 6.21 and 6.22 show the mass spectra extracted from the mobility regions of singly charged APG monomer and APG X-mer (dimer), respectively, of the 1200 UP alkyl polyglucoside sample. It is evident from Figure 6.21 that the concentration of C8 and C10 containing copolymer is markedly lower in plantaren 1200 than in plantacare 818 (Figure 6.17). The polyglycoside distributions in these samples are, however, very similar. Meanwhile, comparision of Figures 6.18 and 6.21 attests that the alkyl chain distribution is narrower for 1200 UP than 818 UP, in agreement with the low C8-C10 content in the former APG. 194
801.2 505.2 667.2 1047.6 1125.5 1153.5 1181.6 885.4 1019.4 589.3 343.2 723.3 315.2 427.3 857.4 639.2 561.2 477.2 695.3 399.2 533.3 371.2 m/z 3000 [G n C m ] + [G n C m ] x (APG + multimers) 2000 [G n C m ] 2 +1 (APG monomers ) 1000 [G n C m ] +1 (APG dimers) 1.81 3.61 5.42 7.22 9.03 10.83 12.64 drift time (ms) 14.44 Figure 6.20 2-D IM-MS plot (m/z vs. drift time) of the 1200 UP dissolved in H2O + 10 % MeOH at 0.05 mg/ml + 1 % NaTFA (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. G₁Na+ # $ G₂Na+ #! G₃Na+ # $!! % G₄Na+ # @ $ @ % @! $ G₅Na+ # $ G₆Na+! @ # 200 300 400 500 600 700 800 900 1000 1100 m/z Figure 6.21 Mass spectrum extracted from the [APG]+ 1 mobility region in the ESI-IM- MS plot of 1200 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)12, # = (CH₂)₁₄, $ = (CH₂)₁₆, and % = (CH₂)₁₈. 195
G₁DI (G₁+G₂)DI 663.3 775.5 719.4 747.4 607 635 691.4 803.5 831 881.5 909.5 937 G₂ DI 1043.5 1071.5 1099.6 (G₁+G₄ & G₂+G₃)DI 1205.6 1233.7 G₃ DI & (G₂+G₄)DI 650 750 850 950 1050 1100 1150 1200 m/z Figure 6.22 Mass spectrum extracted from the [X-mer] +1 mobility region in the ESI-IM- MS plot of 1200 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn. Figure 6.23 shows the 2-D IM-MS plot (m/z vs. drift time) of the 2000 UP APG sample under ESI conditions. Figures 6.24 and 6.24 show the mass spectra extracted for the mobility separated APG monomer and APG dimer with +1 charge, respectively. From these data, it can be concluded that the Cn chain distributions of 818 UP and 2000 UP are comparable, while the corresponding Gn distributions differ slightly, with the latter APG (2000 UP) containing less longer Gn chains. It is also noticed that 2000 UP forms more higher mass dimers (Figure 6.24 vs. Figure 6.19), suggesting that aggregation is promoted with a narrower polyglycoside distribution. 196
m/z 3000 [G n C m ] + [G n C m ] x (APG + multimers) 2000 [G n C m ] 2 +1 (APG monomers) 1000 [G n C m ] +1 (APG dimers) 1.81 3.61 5.42 7.22 9.03 10.83 12.64 drift time (ms) 14.44 Figure 6.23 2-D IM-MS plot (m/z vs. drift time) of the 2000 UP sample dissolved in H2O + 10 % MeOH at 0.05 mg/ml + 1 % NaTFA (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. Figure 6.24 Mass spectrum extracted from the [APG] +1 mobility region in the ESI-IM- MS plot of 2000 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn, and the number of alkyl carbons is labeled by! = (CH₂)₈, @ = (CH₂)12, # = (CH₂)₁₄, $ = (CH₂)₁₆, and % = (CH₂)₁₈. 197
G₁DI 747.4 775.5 635 663.3 691.4 719.4 607 (G₁+G₂)DI 803.5 825.5 853.5 881.6 909.6 959.5 987.5 G₂ DI 1015.6 1043.5 1093.5 (G₁+G₄ & G₂+G₃)DI 1121.5 1149.6 600 700 800 900 1000 1100 m/z Figure 6.25 Mass spectrum extracted from the [X-mer] +1 mobility region in the ESI-IM- MS plot of 2000 UP alkyl polyglucoside. The number of glucose units for each ion is labeled by Gn. Using the mobility separated dimer ions, it is possible to evaluate their yield as a function of the APG concentration. This is illustrated in Figure 6.26 for plantacare 818. Increasing the concentration of this APG in aqueous solution being electrosprayed from 0.001 to 0.1 mg/ml causes a linear increase in the total ion current of its dimer ions. Such dependence provides evidence that the dimers are already present in the aqueous solution and are gently transferred into the gas phase during ESI. The mass spectra extracted from the mobility regions of the higher charge states are uninterpretable, because they contain a large number of overlapping monomers and higher order aggregates. The total ion intensities in these regions can, nevertheless, be 198
used to assess the aggregation (micelle formation) properties of the APGs. (See Appendix for a representative spectrum and total ion intensity data.) 2500 Conc. dependence Total current of dimer (arb.u.) 2000 1500 1000 500 0 y = 21833x + 9.4548 R² = 0.9998 0 0.02 0.04 0.06 0.08 0.1 0.12 APG concentration (mg/ml) Figure 6.26 Total ion intensities of the dimer ions detected from the 818 UP sample, plotted against the corresponding APG concentrations. The drift times of APGs with the same Gn but different Cn chains are similar. For example, the drift times of G₁C₈Na+, G₁C₁₀Na+, and G₁C₁₂Na+ are equal to 1.35 ms, 1.40 ms, and 1.44 ms, respectively. Increasing the alkyl chain length affects the drift time less than increasing the number of glucose units. For example, the drift times of G1C12Na + and G2C12Na+ are 1.44 ms and 1.62 ms, respectively. These trends can be understood by considering that adding an alkyl repeat unit (28 Da) increases the mass and collision cross-section less than adding a glycoside repeat unit (162 Da). The drift times 199
of dimer ions also increase with mass. For example, the dimer ions at m/z 635, 663, and 719 ions have drift times of 2.80 ms, 2.98 ms, and 3.34 ms, respectively. (See Appendix for a list of experimental collision cross-sections.) As mentioned, the IM resolution of the instrument used (Synapt) enabled the separation of APG monomer and dimer ions with +1 charge (Na + ). The IM bands of the higher charge states give very complex and uninterpretable spectra because of the superposition of longer APG ions with aggregates (micelles) of many different sizes. For this reason and in order to obtain more information on the APG components in higher charge states, a monodisperse APG, viz. G2C12, was analyzed under the same conditions. m/z [X-mer]+⁷ [X-mer]+³ 3000 2000 [X-mer]+⁶ [X-mer]+⁵ [X-mer]+⁴ [X-mer]+² [G₂C₁₂+X-mer]+¹ 1000 1.81 3.61 5.42 7.22 9.03 10.83 12.64 drift time (ms) 14.44 16.25 Figure 6.27 2-D IM-MS plot (m/z vs. drift time) of n-dodecyl-α-d-maltoside (α-c₁₂g₂) dissolved in H2O + 10 % MeOH at 0.05 mg/ml + 1 % NaTFA (both v/v %), acquired with the Synapt Q/ToF mass spectrometer. 200
2-mer Na+ 1043.5513 G₂C₁₂ Na+ 533.2584 3-mer Na+ 1553.8842 4-mer Na+ 2064.199 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 m/z Figure 6.28 Mass spectrum extracted from the +1 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α-c₁₂g₂). The APG with the composition G2C12, viz. N-dodecyl-α-D-maltoside (α-c₁₂g₂), is available in high purity. Its MALDI-MS spectrum, acquired under the the same conditions as the MALDI spectra of the other APG samples (section 6.3.1), shows one ion at m/z 533 which corresponds to sodiated C₁₂G₂. For the ESI experiments, a solution of NaTFA salt (0.01 mg/ml) was mixed with α-c₁₂g₂ solution (0.05 mg/ml in H2O + 10 % MeOH) in the ratio 1:100 (v : v %). In the ESI mass spectrum obtained from this sample, C₁₂G₂Na+ (m/z 533) and C₁₂G₂K+ (m/z 549) are detected. (See Appendix) Other ions seen are aggregates and micelles ions from the 2-mer to the 53-mer with +1 until +7 charges. IM spectrometry was used to separate the micelles ions. Figure 6.27 shows the 2-D IM-MS plot (m/z vs. drift time) of the n-dodecyl-α-d-maltoside (α-c₁₂g₂) sample. 201
3594.7395 12-mer +³ 2063.9896 13-mer +³ 14-mer +³ 2234.0729 2404.15623 15-mer +³ 2574.2396 11-mer +³ 1893.9063 16-mer +³ 2744.3229 17-mer +³ 2914.4062 18-mer +³ 1700 1850 2000 2150 2300 2450 2600 2750 2900 3050 m/z Figure 6.29 Mass spectrum extracted from the +3 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α- G₂C₁₂). 29-mer +⁵ 28-mer +⁵ 2982.4372 31-mer +⁵ 2880.3895 3186.5395 27-mer +⁵ 30-mer +⁵ 2778.3395 3084.4895 26-mer +⁵ 32-mer +⁵ 25-mer +⁵ 2574.2396 2676.2895 3288.5895 33-mer +⁵ 35-mer +⁵ 3390.6395 36-mer +⁵ 34-mer +⁵ 3492.6895 3696.7895 37-mer +⁵ 38-mer +⁵ 2350 2500 2650 2800 2950 3100 3250 3400 3550 3700 3850 m/z Figure 6.30 Mass spectrum extracted from the +5 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α-c₁₂g₂). 202
Figure 6.28 shows the mass spectrum extracted from the band of singly charged ions formed by ESI of the n-dodecyl-α-d-maltoside (α-c₁₂g₂) sample. These ions are moving slowly and have relatively long drift times (among ions of similar m/z). They include the sodiated monomer C₁₂G₂Na+ at m/z 533.2584, 2-mer at m/z 1043.5523, 3-mer at m/z 1553.8842, and 4-mer at m/z 2064.199 plus the corresponding potassiated species in much lower abundances. The mass difference between the singly charged n-mer and n+1- mer is 510 Da which is the mass of C₁₂G₂ without the sodium ion. The ions detected in the +2 band range from the sodiated 6-mer ion at m/z 1555.4216 until the sodiated 11-mer at m/z 2830.5078 and have a repeat unit of 510/2-255 m/z units (See Appendix). Figure 6.29 shows the mass spectrum extracted from the band of triply charged ions generated from n-dodecyl-α-d-maltoside (α-c₁₂g₂). The +3 ions detected encompass 11-mer ion at m/z 1894.2844 until the 18-mer ion at m/z 3086.734 with adjacent n-mers appearing every 170 m/z units. The quadruply charged ions detected start at the 16-mer and reach the 27-mer, appearing in intervals of 510/4=127.5 m/z units (See Appendix). Figure 6.30 shows the mass spectrum of the IM band containing the +5 ions from n-dodecyl-α-dmaltoside (α-c₁₂g₂). Here, the 25-mer until the 38-mer are observed with a repeat unit of 510/5=102 m/z units. Finally, in the mass spectra extracted from charge states +6 and +7 aggregates with 34-46 and 47-53 APG monomers, respectively are detected (See Appendix). The results for n-dodecyl-α-d-maltoside, provide clear and strong evidence that APGs aggregate to form micelles in aqueous solutions. 203
m/z 2.98 ms [G₁C₁₂+G₁C₈] Na+ (G₁C₁₀)₂Na+ 600 400 200 1.35 ms G₁C₁₂Na+ G₁C₁₀Na+ G₁C₈Na+ 0.9 1.81 [G₁C₈Na]+ [G₁C₁₂Na]+ 315.2004 371.2668 2.71 3.61 4.51 5.42 drift time (ms) 6.32 [G₁C₁₀Na]+ 343.2289 [G₁C₁₂+G₁C₈] Na+ (G₁C₁₀)₂Na+ 663.4822 250 350 450 550 650 750 850 m/z Figure 6.31 ESI-MS 2 spectrum of the APG dimer ion at m/z 663 (from 818 UP) at energy of 4 ev (trap collision cell), followed by IM separation of the MS 2 products. The 2-D IM- MS 2 plot is shown on the top and the extracted spectrum on the bottom. The sample was dissolved in water at 0.05 mg/ml plus 1 % (v %) MeOH and the data were acquired with the Synapt Q/ToF mass spectrometer. 204
m/z 800 3.34 ms [2(G₁ C₁₀)+Na] + 600 1.35 ms 400 G₁C₁₂Na+ 200 0.9 1.35 1.81 2.26 [G₁C₁₂Na]+ 2.71 3.16 3.61 4.06 4.51 4.96 drift time (ms) 371.2455 [2(G₁C₁₂)] Na+ 719.5396 100 200 300 400 500 600 700 800 900 m/z Figure 6.32 2-D IM-MS 2 plot (top) and extracted spectrum (bottom) of the APG dimer ion at m/z 719 from 1200 UP. The collision energy was 10 ev. The binding mode in the APG aggregates was investigated further by IM-MS 2 experiments on dimer ions. In these experiments, select dimer ions were collisionally 205
activated to fragment and the MS 2 products formed this way were separated by their ion mobilities before ToF mass analysis. The IM-MS 2 results for the m/z 663 dimer, which fragmented efficiently at a collision energy of 4 ev, are shown in Figure 6.31. The dimer gives rise to fragments at 371 (G₁C₁₂Na+), 343 (G₁C₁₀Na+), and 315 (G₁C₈Na+) which means that m/z 663 is composed of two isomeric dimers, i.e. [G₁C₁₂+G₁C8]Na+ and [G₁C₁₀+G₁C₁₀]Na+. The drift times of the dimer ions and their monomeric fragments are 2.98 ms and 1.35 ms, respectively. (See Appendix for the corresponding drift time distributions.) The MS 2 spectrum of the dimer at m/z 719 (Figure 6.32) which drifts at 3.34 ms, acquired at the relatively low collision energy of 10 ev, contains only one fragment at m/z 371 (G₁C₁₂Na+) which drifts at 1.35 ms. Hence, m/z 719 is an aggregate of two G₁C₁₂ units only plus one Na + ion (371+371+23=719 Da). The MS 2 spectrum of the m/z 635 dimer (Figure 6.33) which drifts at 2.80 ms, analyzed also at the of 10 ev, contains ions at m/z 343 (G₁C₁₀Na+) and 315 (G₁C₈Na+), both having drift times equal to 1.35 ms; this means that m/z 635 is a sodiated aggregate of G₁C₁₀ and G₁C₈ [343+315+23=635 Da]. The monomeric APG units associate by hydrogen bonding to form dimers with the molecular weight of both APG units, which are ionized by Na + adduction upon ESI. Because these aggregates are together by noncovalent interactions, low collision energies are sufficient to break them into the monomers. The ESI-MS and MS 2 spectra further reveal that both homodimers (identical alkyl groups), like those at m/z 635 and 663, and heterodimers, like m/z 719 are present in the solution being electrosprayed. Such aggregation agrees well with the micelle-forming properties of APGs. The drift time distributions of the dimer ions (See Appendix) show single peaks, 206
consistent with only one structure for the dimers with one composition (m/z 719 and 635) and very similar shapes and collision cross-sections for the dimers containing isomeric components (m/z 663). 1000 2.80 ms (G₁ C₈+ G₁ C₁₀) Na+ 500 1.35 ms G₁C₁₂Na+ G₁C₈Na+ 1.0 1.50 2.0 2.50 3.0 3.50 4.0 4.50 drift time (ms) [G₁C₈Na]+ 315.1939 [G₁C₁₀Na]+ 343.2289 [G₁C₁₂+G₁C₈] Na+ 635.4482 120 160 200 240 280 320 360 400 440 480 520 560 600 m/z Figure 6.33 2-D IM-MS 2 plot (top) and extracted spectrum (bottom) of the APG dimer ion at m/z 635 from 2000 UP. The collision energy was 10 ev. 207
818 UP Sample 1.04 1.44 1.97 2.60 1200 UP Sample 1.95 2.60 2000 UP Sample 1.02 1.44 1.97 2.62 0.20 0.60 1.00 1.40 1.80 2.20 2.60 Time (min) Figure 6.34 UPLC MS chromatogram (TIC vs. time) of APG samples. Gradient elution as percentage of mobile phase B [methanol/isopropanol (50:50) (v/v %)]: linear increase from 60% to 70% over 1 min, from 70% to 80% over 1 min, and from 80% to 90% over 1 min. The mobile phase flow rate was 250 μl min 1. The retention times of the peak maxima are marked. 6.6 UPLC-MS and UPLC-IM-MS studies of Alkyl Polyglucosides (APGs) To complete the study, the efficacy of UPLC for the separation and identification of APG constituents was also assessed. UPLC which separates according to polarity was also coupled with ion mobility mass spectrometry to analyze further the LC fractions according to the shapes (collision cross-sections) and masses of their components. There are four separated fractions present in the total ion chromatogram of samples 818 UP and 208
2000 UP and two separate fractions present in the total ion chromatogram of sample 1200 UP (Figure 6.34). Each fraction corresponds to different structures. Because a C18 column and reverse phase mode were used, oligomers having the same alkyl chain length and different number of glucose units are eluted together and the alkyl chain length increases with the retention time of the fractions. Figures 6.35 and 6.36 show the LC mass spectra of LC fractions #1, #2, #3, and #4 from the 818 UP sample. Fraction #1 contains oligomers with a C8 group and 1-3 glucose units; traces of G4-G5 oligomers (with a C8 chain) are barely above noise level. Fractions #2, #3, and #4 include oligomers with only C10, C12, and C14 groups, respectively, which also contain from 1 to 3 glucose units (See Appendix for the corresponding LC-MS and LC-IM-MS data). In each fraction, dimers are detected, such as m/z 663 in fraction #2 and m/z 719 in fraction #3. These coelute with the monomers at the same retention times because they contain the same proportion of hydrophobic vs. hydrophilic substituents. For example, the dimer at m/z 663 (G1C10)2, is eluted at 1.44 min, together with the G1C10 (m/z 343) monomer because both have the same Gn/Cn molar (or weight) ratio. 209
393.0974 377.1118 m/z 1000 [G5C8]+¹ [G4C8]+¹ [G3C8]+¹ G1 DI 500 [G2C8]+¹ [G1C8]+¹ 1.0 2.0 3.0 4.0 5.0 drift time (ms) 6.0 7.0 8.0 [G₁C₈Na]+ 315.1549 Fraction #1 GnC₈ [G₁C₈H]+ 293.1835 [G₂C₈H]+ 455.1960 [G 1 DI]+ [G₂C₈Na]+ 477.1831 607.3065 [G₃C₈Na]+ [G 4 C₈Na]+ 801.2390 639.2303 275 375 475 575 675 775 875 975 m/z Figure 6.35 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #1 from the 818 UP APG sample. 210
421.1060 405.1469 m/z 1000 [G4C10]+¹ [G3C10]+¹ G1 DI 500 [G2C10]+¹ [G1C10]+¹ 1.0 2.0 3.0 4.0 5.0 drift time (ms) 6.0 7.0 8.0 [G₁C₁₀Na]+ 343.1857 Fraction #2 GnC 10 [G₂C₁₀Na]+ 505.2041 [G 1 DI]+ 663.3617 275 375 475 575 675 775 875 975 m/z Figure 6.36 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #2 from the 818 UP APG sample. 211
449.1507 433.1686 m/z 1000 [G3C12]+¹ G1 DI 500 [G2C12]+¹ [G1C12]+¹ 1.0 2.0 3.0 4.0 5.0 drift time (ms) 6.0 7.0 8.0 [G₁C₁₂Na]+ 371.2073 [G₁C₁₂H]+ 349.2292 Fraction #3 GnC₁₂ [G₂C₁₂H]+ 511.2568 [G₂C₁₂Na]+ 533.2261 [G 1 DI]+ 719.4035 275 375 475 575 675 775 875 975 m/z Figure 6.37 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #3 from the 818 UP APG sample. 212
477.11590 461.2028 m/z 1000 G1 DI 500 [G2C14]+¹ [G1C14]+¹ 1.0 2.0 3.0 4.0 5.0 drift time (ms) 6.0 7.0 8.0 [G₁C₁₄Na]+ 399.2390 Fraction #4 GnC₁₄ [G₁C₁₄H]+ 377.2547 [G₂C₁₄Na]+ 561.2462 [G 1 DI]+ 775.4426 275 375 475 575 675 775 875 975 m/z Figure 6.38 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #4 from the 818 UP APG sample. 213
The coelution of dimers and monomers causes LC peak broadening which reduces the resolution typically achievable under UPLC conditions. Further, longer Gn chains which were clearly observed in MALDI-MS and ESI-IM-MS spectra, are not detected in the LC-MS spectra, suggesting permanent retention (or precipitation on the column) of APG components with too many polar OH groups. The UPLC-MS results agree with the results from MALDI-MS and ESI-MS experiments, substantiating that 1200 UP sample has barely any APG components with C8 and C10 alkyl chains. Based on the UPLC-MS chromatogram, the amount of C8 blocks in 818 UP appears to be higher than in 2000 UP, whereas the amount of C12 blocks in 818 UP is lower than in 2000 UP. For the UPLC-IM-MS experiments, the APG samples were prepared in the same manner as for LC-MS. The sample solution was introduced into UPLC column and the eluates into the ESI source under positive ion mode. The ions formed from each fraction were separated by their ion mobilities before ToF mass analysis. Figures 6.35 and 6.36 show the LC-IM-MS plots of LC fractions #1, #2, #3, and #4 from the 818 UP sample. Single bands are observed for all APG components and dimers, consistent with one architecture. The other two APGs show very similar results (reproduced in the Appendix). 6.7 Conclusions The finding of this study can be summarized as follows: (1) The compositions of APGs can be rapidly surveyed by MALDI-MS. (2) ESI leads to more complex spectra due to micelle (aggregate) formation. 214
(3) MS 2 confirms the noncovalent binding in the micelles and the glycoside connectivity in the APGs. (4) LC fractionates the APGs based on the length of their alkyl substituents. Dimer / micelle formation in the solutions being fractionated decrease the LC resolution. Longer glycoside chains are not observed in the LC-MS spectra, possibly due to retention / precipitation on the column. (5) IM-MS separates the APGs both by glycoside and alkyl chain length. Longer glycoside chains and traces of longer alkyl chains are detected, whereas they were below noise level in LC-MS. (6) IM-MS provide a more sensitive and more comprehensive (and faster) characterization and also is suitable for evaluating and comparing the micelle forming properties of different APGs. 215
CHAPTER VII SUMMARY Mass spectrometry is a powerful technique that can be used successfully for the study, characterization, and identification not only of small molecules with simple structures, but also of large and more complex compounds, like polymers, proteins, and new materials. The work described in this dissertation has shown the capabilities resulting from using different ion sources and mass analyzers, multiple stages of mass spectrometry, different activation methods to cause ion fragmentation, and hyphenated methods combining mass analysis with separation techniques, like IM spectrometry and UPLC. These capabilities were demonstrated with the analysis of hydrophilic, hydrophobic, and amphiphilic compounds, including peptides, polymers, hybrid materials, and surfactants. Chapter IV reported the complete characterization of two hybrid materials (PtBA- VG2 and PAA-VG2) and its polymer (PAA) and peptide (VG2) components using MALDI-MS, ESI-MS, and IM-MS in positive and negative ion modes. The MALDI-MS and ESI-MS experiments confirmed the formation of hybrid material containing one block of PAA (or PtBA) and VG2 peptide, and the IM-MS results provided strong evidence that multiple PAA-VG2 blocks were also generated. CCS determination and 216
molecular modeling further showed that PAA-VG2 has a macrocyclic structure with alltriazine coupling groups between the PAA and VG2 blocks and not a linear, alkyne/azide terminated structure. MALDI-MS, ESI-MS, IM-MS, and tandem mass spectrometry are also applied to characterize independently VG2 and PAA. Chapter V described the full characterization of two peptides (HA and BMP-2) and a hybrid material [BMP-2(HA)2] containing a dendritic polyethylene glycol (PEG) with two branches terminated by HA (bioactive hydroxyapatite binding peptide), and a focal point substituted with BMP-2 (bioactive peptide that mimics the bone morphogenic protein-2). MALDI-MS, ESI-MS, and IM-MS were applied to analyze the purity and composition of the two peptides and tandem mass spectrometry was applied to confirm their sequences. MALDI-MS, ESI-MS, and IM-MS were used to study the BMP-2(HA)2 sample, which formed multiple charge states upon ESI, ranging from +3 to +9. In IM-MS experiments indicated that BMP-2-(HA)₂ has a folded conformation if it carries 3-4 proton charges, but that is unfolds to a more extended conformation after more than 4 charges have been added. This was confirmed using molecular modeling and comparison of measured and calculated CCSs. Chapter VI, the complete characterization of three APG samples supplied by BASF was reported. MS and MS² experiments under MALDI and ESI conditions helped to determine the number of glucose units and the alkyl chain length in the APG samples. The ESI tandem mass spectra in also revealed the connectivity of the polyglycoside (1, 4). APG aggregates with different charges were observed with ESI, originating from the micelles formed by the amphiphilic APGs in the aqueous solutions analyzed. The APG surfactants could be separated according to the number of glucose units and the degree of 217
aggregation by IM-MS. On the other hand, UPLC-MS separated the APG constituents based on their alkyl block lengths. Overall, this dissertation demonstrated the analytical power of multidimentional mass spectrometry methods for the complete characterization of hybrid materials and biodegradable copolymer. 218
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APPENDICES 241
APPENDIX A ADDITONAL DATA Table A1 Table A2 Table A3 Table A4 Table A5 Table A6 Table A7 Table A8 Drift time and collision cross-section data of singly and doubly protonated polyalanine oligomers, H-(Ala)n OH using published CCSHe data from reference 139.247 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of doubly charged ions Figure A1...249 Comparison of published collision cross-sections of the triply protonated polyalanine oligomers formed by ESI with values determined in this study using the calibration plot of doubly charged ions Figure A1...250 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of singly charged ions Figure A1...251 Comparison of published collision cross-sections of the triply protonated polyalanine oligomers formed by ESI with values determined in this study using the calibration plot of singly charged ions Figure A1...252 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot from Figure A2 253 Comparison of published collision cross-sections of the triply protonated polyalanine oligomers formed by ESI with values determined in this study using the calibration plot of Figure A2......254 Drift time and collision cross-section data of singly and doubly protonated polyalanine oligomers, H-(Ala)n OH using published CCSN₂ values from reference 139..255 242
Table A9 Table A10 Table A11 Table A12 Table A13 Table A14 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of polyalanine using CCSN2 values (Figure A3)...257 Comparison of published N2 collision cross-sections of triply protonated polyalanine oligomers formed by ESI with values determined in this study using the calibration plot of Figure A3 using CCSN₂....258 Drift time and collision cross-section data of doubly protonated polyalanine oligomers, H-(Ala)n OH using published CCSHe from reference 139.259 Experimental collision cross-sections of nine doubly and triply protonated PtBAn-VG2 oligomers, derived using the calibration plot of Figure A4...261 Experimental collision cross-sections of twelve doubly and triply protonated PAAn-VG2 oligomers, derived using the calibration plot of Figure A4...263 Total ion current (TIC) of higher charge states (in % of TIC for APG dimers.269 Table A15 Drift times (ms) and collision cross-sections (Å 2 ) of GmCnNa + from 818 UP sample. Drift times were measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8 V. Experimental collision cross-sections of protonated APG ions, derived using the calibration plot of Figure 5.33.269 Figure A1 Figure A2 Plot of corrected drift times (arrival times) against corrected published cross-sections for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A1. The equation in the top corner is from power fitting the doubly charged ions and the equations in the bottom corner is from power fitting the singly charged ions..248 Plot of corrected drift times (arrival times) against corrected published cross- sections for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A1. The equation is for singly and doubly charged ions power fitting together......248 243
Figure A3 Figure A4 Figure A5 Figure A6 Figure A7 Figure A8 Figure A9 Plot of corrected drift times (arrival times) against corrected published cross-sections with N2 gas for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A8. The equation is from power fitting singly and doubly charged ions together...256 Plot of corrected drift times (arrival times) against corrected published cross-sections with He gas for the +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A11. The equation is from power fitting doubly charged ions...260 Plot of calculated collision cross-sections (PA method) for PtBA-VG2 oligomers with linear and cyclic structures (a) +2 or (b) +3 charges, and experimental collision cross-sections of the same oligomers vs. m/z ratio. The Experimental collision cross-sections is from Table A12..262 Plot of calculated collision cross-sections (PA method) for PAA-VG2 oligomers with linear and cyclic structures (a) +2 or (b) +3 charges, and experimental collision cross-sections of the same ions vs. m/z ratio. The Experimental collision cross-sections is from Table A13.264 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 4 proton charges at the following sits: HA1 (K, R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 61 kcal/mol and 905 Ų, respectively. A representative structure is inserted inside the plot.265 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 4 proton charges at the following sits: BMP-2 (K, K; HA1 (R); HA2 (R). The average relative energy and CCS (PA) of all 50 candidate structures are 54 kcal/mol and 923 Ų, respectively. A representative structure is inserted inside the plot..265 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 4 proton charges at the following sits: BMP-2 (K); HA1 (R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 67 kcal/mol and 933 Ų, respectively. A representative structure is inserted inside the plot...266 244
Figure A10 Figure A11 Figure A12 Figure A13 Figure A14 Figure A15 Figure A16 Figure A17 Figure A18 Figure A19 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 5 proton charges at the following sits: BMP-2 (K); HA1 (K, R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 74 kcal/mol and 920 Ų, respectively. A representative structure is inserted inside the plot...266 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 6 proton charges at the following sits: BMP-2 (K, K); Triazoles (N, N); HA1 (R); HA2 (R). The average relative energy and CCS (PA) of all 50 candidate structures are 34 kcal/mol and 785 Ų, respectively. A representative structure is inserted inside the plot...267 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 6 proton charges at the following sits: BMP-2 (K, K); HA1 (K, R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 59 kcal/mol and 908 Ų, respectively. A representative structure is inserted inside the plot...267 Mass spectrum extracted from the +2 band in the ESI-IM-MS plot of 818 UP. See Figure 6.17 for the ESI-IM-MS plot.268 Mass spectrum extracted from the +3 band in the ESI-IM-MS plot of 818 UP. See Figure 6.17 for the ESI-IM-MS plot.268 Mass spectrum extracted for the +2 band in the ESI-IM-MS plot of n-dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot....270 Mass spectrum extracted for the +4 band in the ESI-IM-MS plot of n-dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot....270 Mass spectrum extracted for the +6 band in the ESI-IM-MS plot of n-dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot....271 Mass spectrum extracted for the +7 band in the ESI-IM-MS plot of n-dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot....271 Drift time distributions for APG dimer ions (right) and their monomeric fragments (left). See Figures 6.31-6.32 for the corresponding ESI-IM- MS 2 spectra 272 245
Figure A20 Figure A21 Figure A22 Figure A23 Figure A24 Figure A23 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #3 from the 1200 UP APG sample...273 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #4 from the 1200 UP APG sample...274 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #1 from the 2000 UP APG sample...275 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #2 from the 2000 UP APG sample...276 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #3 from the 2000 UP APG sample...277 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #4 from the 2000 UP APG sample...278 246
Table A1 Drift time and collision cross-section data of singly and doubly protonated polyalanine oligomers, H-(Ala)n OH using published CCSHe data from reference 139. z a n moligmer (Da) m/z td b td' c Ω (Ų) reduced mass d Ω' (Ų) 1 4 302.11 303.20 2.19 2.1655 100 5.06295 506.2957 1 5 373.13 374.14 2.76 2.7327 114 5.10433 581.8936 1 6 444.15 445.16 3.33 3.3003 128 5.13307 657.0324 1 7 515.18 516.18 4.09 4.0580 141 5.15419 726.7402 1 8 586.19 587.20 4.96 4.9258 157 5.17036 811.7468 1 9 657.21 658.22 5.75 5.7138 170 5.18315 881.1353 1 10 728.24 729.25 6.49 6.4519 181 5.19351 940.0256 1 11 799.27 800.28 7.42 7.3801 194 5.20208 1009.2035 1 12 870.29 871.30 8.18 8.1384 206 5.20928 1073.1121 1 13 941.33 942.34 8.98 8.9367 217 5.21542 1131.7465 1 14 1012.37 1013.38 9.90 9.8551 228 5.22072 1190.3237 2 17 1225.42 613.72 3.07 3.0351 265 5.23298 693.3797 2 18 1296.43 649.22 3.34 3.3041 276 5.23618 722.5935 2 19 1367.45 684.74 3.58 3.5431 287 5.23906 751.8055 2 20 1438.76 720.39 3.86 3.8222 297 5.24167 778.3881 2 21 1509.51 755.77 4.11 4.0712 308 5.24402 807.5788 2 22 1580.54 791.28 4.42 4.3803 317 5.24617 831.5173 2 23 1651.55 826.78 4.75 4.7045 327 5.24813 858.0694 2 24 1722.59 862.30 4.98 4.9336 337 5.24994 884.6143 2 25 1793.61 897.82 5.32 5.2758 348 5.2516 913.7785 2 26 1864.64 933.33 5.71 5.6619 358 5.25314 940.3119 a CCS values were taken from reference 139 for the singly and doubly charged ions. b Measured drift time c t D` = t D - [ 1.41 m z 1000 ] d Reduced mass = m ion m N m ion +m N, where m ion and m N are the masses of the polyalanine ion (see Table) and the drift gas molecule (N2, 28 Da), respectively. e obtained via Ω`= Ω*( m ion m N m ion +m N /z) 247
Normalised CCS (Ω ) (Ų) Normalised CCS (Ω ) (Ų) 1400 1200 1000 800 600 400 200 0 y = 402.84x 0.4911 R² = 0.9989 Polyalanine WV=350, HW=8 y = 332.05x 0.5591 R² = 0.9995 0 2 4 6 8 10 12 Corrected drift time (t D ) (ms) PA +1CH PA +2CH Figure A1 Plot of corrected drift times (arrival times) against corrected published crosssections for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A1. The equation in the top corner is from power fitting the doubly charged ions and the equation in the bottom corner is from power fitting the singly charged ions. Polyalanine WV=350, HW=8 1400 1200 1000 800 600 400 200 0 y = 371.22x 0.5165 R² = 0.9498 0 2 4 6 8 10 12 Corrected drift time (t D ) (ms) Figure A2 Plot of corrected drift times (arrival times) against corrected published crosssections for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A1. The equation is from power fitting singly and doubly charged ions together. 248
Table A2 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of doubly charged ions in Figure A1. z m ion (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 9 8569.15 952.128 5.00 4.956 5.2838 884.164 1506 1649-9 10 8570.13 857.013 4.36 4.319 5.2838 826.334 1564 1732-11 11 8571.27 779.206 3.98 3.941 5.2838 789.979 1645 1802-10 13 12367.50 951.345 5.00 4.957 5.2864 884.165 2174 2391-10 14 12368.50 883.466 4.62 4.578 5.2864 850.344 2252 2473-10 15 12369.50 824.635 4.23 4.190 5.2864 814.098 2310 2579-12 16 12370.40 773.150 4.10 4.061 5.2864 801.718 2426 2679-10 17 12372.30 727.784 3.85 3.812 5.2864 777.203 2499 2723-9 18 12374.10 687.448 3.59 3.553 5.2864 750.813 2556 2766-8 19 12375.60 651.348 3.33 3.294 5.2864 723.415 2600 2800-8 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A1 (y=402.84 x 0.4911 ) d exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 249
Table A3 Comparison of published collision cross-sections of triply protonated polyalanine oligomers formed by ESI with values determined in this study using the calibration plot of doubly charged ions in Figure A1. z moligomer (Da) m/z td (ms) td' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 3 1864.64 622.554 3.25 3.215 5.2531 714.821 408 425-4 3 1935.64 646.222 3.52 3.484 5.2546 743.630 425 438-3 3 2006.66 669.896 3.70 3.664 5.2559 762.188 435 452-4 3 2077.71 693.577 3.97 3.933 5.2571 789.213 450 465-3 3 2148.76 717.331 4.24 4.202 5.2583 815.312 465 479-3 3 2219.77 740.932 4.51 4.472 5.2594 840.574 479 490-2 3 2290.75 764.590 4.60 4.561 5.2604 848.785 484 502-4 3 2361.82 788.283 4.87 4.830 5.2613 873.046 498 516-4 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A1 (y=402.84 x 0.4911 ) d exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 250
Table A4 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of singly charged ions in Figure A1. z m ion (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 9 8569.15 952.128 5.00 4.956 5.2838 812.598 1384 1649-19 10 8570.13 857.013 4.36 4.319 5.2838 752.369 1424 1732-22 11 8571.27 779.206 3.98 3.941 5.2838 714.801 1488 1802-21 13 12367.50 951.345 5.00 4.957 5.2865 812.599 1998 2391-20 14 12368.50 883.466 4.62 4.578 5.2865 777.306 2060 2473-20 15 12369.50 824.635 4.23 4.190 5.2865 739.699 2099 2579-23 16 12370.40 773.150 4.10 4.061 5.2865 726.905 2200 2679-22 17 12372.30 727.784 3.85 3.812 5.2865 701.655 2256 2723-21 18 12374.10 687.448 3.59 3.553 5.2865 674.595 2297 2766-20 19 12375.60 651.348 3.33 3.294 5.2865 646.642 2324 2800-20 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A1 (y=332.05 x 0.5591 ) d exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 251
Table A5 Comparison of published collision cross-sections of triply protonated polyalanine formed by ESI with values determined in this study using the calibration plot of singly charged ions in Figure A1. z m n-mer (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 3 1864.64 622.554 3.25 3.215 5.2531 637.903 364 425-17 3 1935.64 646.222 3.52 3.484 5.2546 667.253 381 438-15 3 2006.66 669.896 3.70 3.664 5.2559 686.243 392 452-15 3 2077.71 693.577 3.97 3.933 5.2571 714.012 407 465-14 3 2148.76 717.331 4.24 4.202 5.2583 740.954 423 479-13 3 2219.77 740.932 4.51 4.472 5.2594 767.147 438 490-12 3 2290.75 764.590 4.60 4.561 5.2604 775.684 442 502-13 3 2361.82 788.283 4.87 4.830 5.2613 800.975 457 516-13 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A1 (y=332.05 x 0.5591 ) d exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 252
Table A6 Comparison of published collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot from in Figure A2. z m ion (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 9 8569.15 952.128 5.00 4.956 5.2838 848.573 1445 1649-14 10 8570.13 857.013 4.36 4.319 5.2838 790.301 1496 1732-16 11 8571.27 779.206 3.98 3.941 5.2838 753.775 1569 1802-15 13 12367.50 951.345 5.00 4.957 5.2865 848.574 2087 2391-15 14 12368.50 883.466 4.62 4.578 5.2865 814.470 2157 2473-15 15 12369.50 824.635 4.23 4.190 5.2865 777.998 2208 2579-17 16 12370.40 773.150 4.10 4.061 5.2865 765.559 2317 2679-16 17 12372.30 727.784 3.85 3.812 5.2865 740.960 2383 2723-14 18 12374.10 687.448 3.59 3.553 5.2865 714.522 2433 2766-14 19 12375.60 651.348 3.33 3.294 5.2865 687.126 2470 2800-13 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A2 (y=371.22 x 0.5165 ) d exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 253
Table A7 Comparison of published collision cross-sections of triply protonated polyalanine oligomers formed by ESI values determined in this study using the calibration plot of in Figure A2. z m ion (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 3 1864.64 622.554 3.25 3.215 5.2531 678.544 387.507 425-10 3 1935.64 646.222 3.52 3.484 5.2546 707.334 403.84 438-8 3 2006.66 669.896 3.70 3.664 5.2559 725.912 414.342 452-9 3 2077.71 693.577 3.97 3.933 5.2571 753.006 429.706 465-8 3 2148.76 717.331 4.24 4.202 5.2583 779.218 444.566 479-8 3 2219.77 740.932 4.51 4.472 5.2594 804.630 458.970 490-7 3 2290.75 764.590 4.60 4.561 5.2604 812.899 463.597 502-8 3 2361.82 788.283 4.87 4.830 5.2613 837.354 477.457 516-8 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A2 (y=371.22 x 0.5165 ) d exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 254
Table A8 Drift time and collision cross-section data of singly and doubly protonated polyalanine oligomers, H-(Ala)n OH using published CCSN₂ values from reference 139. z a n m n-mer m/z b t D t D' c Ω reduced Ω' (Da) (Ų) mass d (Ų) 1 4 302.11 303.12 2.19 2.1655 166 5.06295 840.4490 1 5 373.13 374.14 2.76 2.7327 181 5.10433 923.8838 1 6 444.15 445.16 3.33 3.3003 195 5.13307 1000.9477 1 7 515.18 516.18 4.09 4.0580 211 5.15419 1087.5332 1 8 586.19 587.20 4.96 4.9258 228 5.17036 1178.8424 1 9 657.21 658.22 5.75 5.7138 243 5.18315 1259.5051 1 10 728.24 729.25 6.49 6.4519 256 5.19351 1329.5390 1 11 799.27 800.28 7.42 7.3801 271 5.20208 1409.7637 1 12 870.29 871.30 8.18 8.1384 282 5.20928 1469.0175 1 13 941.33 942.34 8.98 8.9367 294 5.21542 1533.3340 1 14 1012.37 1013.38 9.90 9.8551 306 5.22072 1597.5396 2 17 1225.42 613.72 3.07 3.0351 369 5.23298 965.4846 2 18 1296.43 649.22 3.34 3.3041 380 5.23618 994.8751 2 19 1367.45 684.74 3.58 3.5431 393 5.23906 1029.4758 2 20 1438.76 720.39 3.86 3.8222 404 5.24167 1058.8174 2 21 1509.51 755.77 4.11 4.0712 416 5.24402 1090.7557 2 22 1580.54 791.28 4.42 4.3803 428 5.24617 1122.6795 2 23 1651.55 826.78 4.75 4.7045 437 5.24813 1146.7166 2 24 1722.59 862.30 4.98 4.9336 448 5.24994 1175.9858 2 25 1793.61 897.82 5.32 5.2758 458 5.25160 1202.6165 2 26 1864.64 933.33 5.71 5.6619 470 5.25314 1234.4877 a CCSN₂ values were taken from reference 139 for the singly and doubly charged ions. b Measured drift time c t D` = t D - [ 1.41 m z 1000 ] d Reduced mass= m ion m N m ion +m N, where m ion and m N are the masses of the polyalanine ion (see Table) and the drift gas molecule (N2, 28 Da), respectively. e Obtained via Ω`= Ω*( m ion m N m ion +m N /z) 255
Normalised CCS (Ω ) (Ų) 1800 1600 1400 1200 1000 800 600 400 200 0 Polyalanine using Ω N₂ WV=350, HW=8 y = 621.07x 0.3981 R² = 0.9985 0 2 4 6 8 10 12 Corrected drift time (t D ) (ms) PA +1CH PA +2 CH Figure A3 Plot of corrected drift times (arrival times) against corrected published crosssections measured with N2 gas for the +1 and +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A8. The equation is from power fitting singly and doubly charged ions together. 256
Table A9 Comparison of published N2 collision cross-sections different charge states of ubiquitin and cytochrome C with values determined in this study using the calibration plot of polyalanine obtained with CCSN2 values (Figure A3). z m ion (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω' c (Ų) Ω d (Ų) Published Ω (Ų) % error e 7 8568.20 1224.028 7.82 7.771 5.2838 1404.857 1861 1910-3 8 8568.33 1071.041 6.03 5.984 5.2838 1266.067 1917 1990-4 9 8569.15 952.128 5.00 4.957 5.2838 1174.599 2001 2090-4 10 8570.13 857.013 4.36 4.319 5.2838 1111.925 2104 2200-5 11 8571.27 779.206 3.98 3.941 5.2838 1072.101 2232 2340-5 12 8573.16 714.430 3.72 3.682 5.2838 1043.550 2370 2380-0 13 8575.26 659.635 3.33 3.294 5.2838 998.240 2456 2600-6 13 12367.49 951.345 5.00 4.957 5.2865 1174.600 2888 3080-7 14 12368.52 883.466 4.62 4.578 5.2865 1138.044 3014 3200-6 15 12369.53 824.635 4.23 4.190 5.2865 1098.560 3117 3330-7 16 12370.40 773.150 4.10 4.061 5.2865 1084.997 3284 3450-5 17 12372.33 727.784 3.85 3.812 5.2865 1058.025 3402 3600-6 18 12374.06 687.448 3.59 3.553 5.2865 1028.807 3503 3670-5 19 12375.61 651.348 3.33 3.294 5.2865 998.268 3588 3790-6 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A3 (y=621.07 x 0.3981 ) d Exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 257
Table A10 Comparison of published N2 collision cross-sections of triply protonated polyalanine oligomers formed by ESI with values determined in this study using the calibration plot of Figure A3 using CCSN₂ z m n-mer (Da) m/z t D (ms) t D' a (ms) Reduced mass b Ω c (Ų) Ω d (Ų) Published Ω (Ų) 3 1864.64 622.554 3.25 3.215 5.2531 988.643 565 576-2 3 1935.64 646.222 3.52 3.484 5.2546 1020.821 583 592-2 3 2006.66 669.896 3.70 3.664 5.2559 1041.425 594 606-2 3 2077.71 693.577 3.97 3.933 5.2571 1071.259 611 621-2 3 2148.76 717.331 4.24 4.202 5.2583 1099.887 628 634-1 3 2219.77 740.932 4.51 4.472 5.2594 1127.433 643 649-1 3 2290.75 764.590 4.60 4.561 5.2604 1136.352 648 666-3 3 2361.82 788.283 4.87 4.830 5.2613 1162.612 663 674-2 a t D` = t D - [ 1.41 m z 1000 ] b Reduced mass= m ion m N m ion +m N c Ω` obtained from curve in Figure A3 (y=621.07 x 0.3981 ) d Exp Ω=Ω` *(z/ m ion m N m ion +m N e % error= [(Exp Ω-Published Ω)/ Exp Ω]*100 % error e 258
Table A11 Drift time and collision cross-section data of doubly protonated polyalanine oligomers, H-(Ala)n OH using published CCSHe values from reference 139. z n m n-mer (Da) m/z td a (ms) td' b Ω (Ų) reduced mass c Ω' d (Ų) 2 17 1225.42 613.72 3.07 3.0351 265 5.23298 693.370 2 18 1296.43 649.22 3.34 3.3041 276 5.23618 722.593 2 19 1367.45 684.74 3.58 3.5431 287 5.23906 751.806 2 20 1438.76 720.39 3.86 3.8222 297 5.24167 778.388 2 21 1509.51 755.77 4.11 4.0712 308 5.24402 807.579 2 22 1580.54 791.28 4.42 4.3803 317 5.24617 831.517 2 23 1651.55 826.78 4.75 4.7045 327 5.24813 858.069 2 24 1722.59 862.30 4.98 4.9336 337 5.24994 884.614 2 25 1793.61 897.82 5.32 5.2758 348 5.25160 913.778 2 26 1864.64 933.33 5.71 5.6619 358 5.25314 940.312 a Measured drift time b t D` = t D - [ 1.41 m z 1000 ] c Reduced mass = m ion m N m ion +m N, where m ion and m N are the masses of the polyalanine ion (see Table) and the drift gas molecule (N2, 28 Da), respectively. d Obtained via Ω`= Ω*( m ion m N m ion +m N /z) 259
Figure A4 Plot of corrected drift times (arrival times) against corrected published crosssections measured with He gas for the +2 ions formed by ESI of polyalanine. Drift times were measured at a wave velocity of 350 m/s and a wave height of 8 V. Normalised CCSs were taken from Table A11. The equation is from power fitting doubly charged ions. 260
Table A12 Experimental collision cross-sections of nine doubly and triply protonated PtBAn-VG2 oligomers, derived using the calibration plot of Figure A4. z n m n-mer (Da) m/z t D (ms) t D` (ms) Reduced mass Ω' (Ų) Ω (Ų) 2 4 1850.09 926.053 5.50 5.5290 5.2528 932.926 355 2 6 2106.35 1054.185 6.41 6.4411 5.2576 1005.568 383 2 7 2234.48 1118.246 7.05 7.0820 5.2596 1053.526 401 2 8 2362.54 1182.280 7.70 7.7330 5.2614 1100.017 418 2 10 2618.70 1310.359 8.72 8.7548 5.2644 1169.148 444 3 5 a 2194.30 732.443 3.08 3.1057 5.2590 702.795 401 3 6 a 2322.48 775.168 3.33 3.3564 5.2608 730.115 416 3 7 a 2450.51 817.844 3.59 3.6172 5.2625 757.441 432 3 9 a 2706.67 903.228 4.00 4.0286 5.2653 798.594 455 a the triply protonated oligomers also contain 3 hydrolyzed PAA repeat units. 261
(a) PtBA n -VG 2 (+2 charge) 600 CCS 550 500 450 400 4 n= 6 7 8 10 11 12 PtBA-VG2 Cyclic calc PtBA-V2 Linear calc PtBA-V2 exp 350 300 900 1000 1100 1200 1300 1400 1500 m/z (b) PtBA n PAA₃-VG₂ (+3 charge) CCS 550 500 450 n= 5 6 7 9 PtBA-VG2 Cyclic calc PtBA-VG2 Linear calc PtBA-VG2 exp 400 350 700 750 800 850 900 950 m/z Figure A5 Plot of calculated collision cross-sections (PA method) for PtBA-VG2 oligomers with linear and cyclic structures (a) +2 or (b) +3 charges, and experimental collision cross-sections of the same oligomers vs. m/z ratio. The Experimental collision cross-sections is from Table A12. 262
Table A13 Experimental collision cross-sections of twelve doubly and triply protonated PAAn-VG2 oligomers, derived using the calibration plot of Figure A4. z n m n-mer (Da) m/z t D (ms) t D` (ms) Reduced mass Ω' (Ų) Ω (Ų) 2 7 1841.93 921.971 4.75 4.7790 5.2527 868.466 331 2 8 1912.76 957.387 4.89 4.9195 5.2541 880.920 335 2 10 2057.84 1029.926 5.47 5.4957 5.2568 930.159 354 2 12 2201.88 1101.950 5.96 5.9918 5.2591 970.489 369 2 14 2346.13 1174.072 6.28 6.3129 5.2611 995.688 379 2 15 2417.94 1209.976 6.39 6.4184 5.2621 1003.837 382 2 16 2490.88 1246.447 6.93 6.9639 5.2629 1044.868 497 3 10 2058.06 687.027 2.61 2.6348 5.2568 648.279 370 3 12 2201.84 734.954 2.69 2.7157 5.2591 657.979 375 3 14 2346.04 783.022 2.82 2.8466 5.2611 673.365 384 3 15 2417.89 806.971 3.03 3.0570 5.2621 697.366 398 3 16 2490.08 831.034 3.08 3.1074 5.2629 702.991 401 263
(a) PAA n -VG₂ (+2 charge) CCS 500 450 400 7 n= 8 10 12 14 15 16 PAA-VG2 Cyclic Calc PAA-VG2 exp PAA-VG2 Linear Calc 350 300 900 1000 1100 1200 1300 m/z (b) PAA n -VG₂ (+3 charge) CCS 550 500 450 n= 10 12 14 15 16 PAA-VG2 Cyclic calc PAA-V2 Linear calc PAA-VG2 exp 400 350 650 700 750 800 850 m/z Figure A6 Plot of calculated collision cross-sections (PA method) for PAA-VG2 oligomers with linear and cyclic structures (a) +2 or (b) +3 charges, and experimental collision cross-sections of the same ions vs. m/z ratio. The Experimental collision crosssections is from Table A13. 264
Collision Cross-section (Ų) 1400 1200 Collision Cross-section (Ų) 1000 800 600 400 905 Ų 200 0 0 50 100 150 200 250 300 350 Relative Potential Energy (kcal/mol) Figure A7 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 4 proton charges at the following sits: HA1 (K, R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 61 kcal/mol and 905 Ų, respectively. A representative structure is inserted inside the plot. 1400 1200 1000 923 Ų 800 600 400 200 0 0 50 100 150 200 250 300 350 Relative Potential Energy (kcal/mol) Figure A8 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 4 proton charges at the following sits: BMP-2 (K, K; HA1 (R); HA2 (R). The average relative energy and CCS (PA) of all 50 candidate structures are 54 kcal/mol and 923 Ų, respectively. A representative structure is inserted inside the plot. 265
1400 1200 1000 Collision Cross-section (Ų) Collision cross-section (Ų) 800 600 933 Ų 400 200 0 0 50 100 150 200 250 300 350 Relative Potential Energy (kcal/mol) Figure A9 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 4 proton charges at the following sits: BMP-2 (K); HA1 (R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 67 kcal/mol and 933 Ų, respectively. A representative structure is inserted inside the plot. 1400 1200 1000 800 920 Ų 600 400 200 0 0 50 100 150 200 250 300 350 Relative Potential Energy (kcal/mol) Figure A10 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 5 proton charges at the following sits: BMP-2 (K); HA1 (K, R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 74 kcal/mol and 920 Ų, respectively. A representative structure is inserted inside the plot. 266
Collision Cross-section (Ų) 1400 1200 Collision Cross-section (Ų) 1000 800 600 400 785 Ų 200 0 0 50 100 150 200 250 Relative Potential Energy (kcal/mol) Figure A11 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 6 proton charges at the following sits: BMP-2 (K, K); Triazoles (N, N); HA1 (R); HA2 (R). The average relative energy and CCS (PA) of all 50 candidate structures are 34 kcal/mol and 785 Ų, respectively. A representative structure is inserted inside the plot. 1400 1200 1000 800 908 Ų 600 400 200 0 0 50 100 150 200 250 Relative Potential Energy (kcal/mol) Figure A12 Plot of collision cross-section vs. relative energy for 50 energy minimized structures of BMP-2(HA)2 with + 6 proton charges at the following sits: BMP-2 (K, K); HA1 (K, R); HA2 (K, R). The average relative energy and CCS (PA) of all 50 candidate structures are 59 kcal/mol and 908 Ų, respectively. A representative structure is inserted inside the plot. 267
0.05 818up+1%na 0.01ng/ml) hw 8 vw 200 AA-07012013-818UP_IM_3_dt_03 41 (3.610) Cm (13:90) 924.3961974.9049 1074.9537 100 TOF MS ES+ 22 924.3738924.8772 1024.9191 1514.1483 1741.7656 1274.9830 1581.1335 1635.2269 1088.7834 1329.9556 1513.6045 1579.7003 1124.9839 1486.1232 1702.2145 1861.7795 875.3615 1251.3716 1475.9727 1422.9933 1797.2391 1900.8544 1929.3322 1250.8381 1945.9135 1957.4683 2076.5225 % 0 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250 2300 2350 2400 2450 m/z Figure A13 Mass spectrum extracted from the +2 band in the ESI-IM-MS plot of 818 UP. See Figure 6.17 for the ESI-IM-MS plot. 0.05 818up+1%na 0.01ng/ml) hw 8 vw 200 AA-07012013-818UP_IM_3_dt_04 45 (3.971) Cm (1:102) 100 2182.5959 2240.9141 TOF MS ES+ 16 2161.1155 2098.6582 1929.5262 2066.4771 2007.5713 2336.9851 2428.4688 2474.2930 2533.7302 % 0 m/z 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 Figure A14 Mass spectrum extracted from the +3 band in the ESI-IM-MS plot of 818 UP. See Figure 6.17 for the ESI-IM-MS plot. 268
Table A14 Total ion current (TIC) of higher charge states (in % of TIC for APG dimers. z 818 UP 1200 UP 2000 UP +2 16.2 % 15.4 % 19.03 % +3 21.4 % 18.69 % 25.37 % Table A15 Drift times (ms) and collision cross-sections (Å 2 ) of GmCnNa + from 818 UP sample. Drift times were measured at a traveling wave velocity of 250 m/s and a traveling wave height of 8 V. Experimental collision cross-sections of protonated APG ions, derived using the calibration plot of Figure 5.33. C8 C10 C12 C14 G1 td 1.26 1.35 1.53 1.62 CCS 126 129 137 141 G2 td 2.26 2.53 2.71 2.98 CCS 164 173 179 187 G3 td 3.34 3.61 3.88 3.97 CCS 197 204 211 214 G4 td 4.24 4.33 4.78 5.05 CCS 220 222 233 240 269
7-mer +² 1808.8646 8-mer +² 2063.9896 9-mer +² 2319.1146 10-mer +² 2575.2396 6-mer +² 1553.7397 11-mer +² 1300 1500 1700 1900 2100 2300 2500 2700 2900 m/z Figure A15 Mass spectrum extracted from the +2 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot. 20-mer +⁴ 21-mer +⁴ 19-mer +⁴ 2574.2396 2446.6771 2701.8020 18-mer +⁴ 2319.1146 22-mer +⁴ 2829.3645 23-mer +⁴ 2956.9270 16-mer +⁴ 17-mer +⁴ 2191.5521 24-mer +⁴ 3084.4895 25-mer +⁴ 3212.0520 26-mer +⁴ 3339.6145 27-mer +⁴ 28-mer +⁴ 2000 2200 2400 2600 2800 3000 3200 3400 3600 m/z Figure A16 Mass spectrum extracted from the +4 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot. 270
40-mer +⁶ 38-mer +⁶ 3424.6561 3254.5728 39-mer +⁶ 37-mer +⁶ 3339.6145 35-mer +⁶ 3169.5312 2999.4478 36-mer +⁶ 3084.4895 41-mer +⁶ 3509.6978 42-mer +⁶ 3594.7395 43-mer +⁶ 45-mer +⁶ 3679.7811 44-mer +⁶ 3764.8228 2849.8644 46-mer +⁶ 34-mer +⁶ 2914.4062 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 m/z Figure A17 Mass spectrum extracted from the +6 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot. 51-mer +⁷ 3740.5252 47-mer +⁷ 3448.9538 48-mer +⁷ 49-mer +⁷ 3521.8466 3594.7395 50-mer +⁷ 3667.6323 52-mer +⁷ 3813.4180 53-mer +⁷ 3886.3109 3300 3340 3380 3420 3460 3500 3540 3580 3620 3680 3700 3740 3780 3820 3860 m/z Figure A18 Mass spectrum extracted from the +7 band in the ESI-IM-MS plot of n- dodecyl-α-d-maltoside (α-c₁₂g₂). See Figure 6.26 for the ESI-IM-MS plot. 271
m/z 315 + m/z 343 1.35 m/z 635 Figure 6.33 2.80 1.35 m/z 315 + m/z 343 + m/z 371 m/z 663 2.98 Figure 6.31 1.35 m/z 371 m/z 719 3.34 Figure 6.32 1.00 1.40 1.80 2.20 2.60 3.00 3.40 3.80 drift time (ms) Figure A19 Drift time distributions for APG dimer ions (right) and their monomeric fragments (left). See Figures 6.31-6.32 for the corresponding ESI-IM-MS 2 spectra. 272
449.1429 433.1686 m/z 1000 G1DI 500 [G2C12]+¹ [G1C12]+¹ 1.0 2.0 3.0 4.0 5.0 6.0 7.0 drift time (ms) [G₁C₁₂Na]+ 371.2073 [G₁C₁₂H]+ 349.2292 Fraction #3 GnC₁₂ [G₂C₁₂H]+ 511.2568 [G₂C₁₂Na]+ 533.2261 [G 1 DI]+ 719.4035 275 375 475 575 675 775 875 975 m/z Figure A20 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #3 from the 1200 UP APG sample. 273
477.11750 461.1949 m/z 1000 G1DI 500 [G2C14]+¹ [G1C14]+¹ 1.0 2.0 3.0 4.0 5.0 6.0 7.0 drift time (ms) [G₁C₁₄Na]+ 399.2390 Fraction #4 GnC₁₄ [G₁C₁₄H]+ 377.2618 [G₂C₁₄H]+ 539.2902 [G 1 DI]+ 775.4429 [G₂C₁₄Na]+ 561.2549 275 375 475 575 675 775 875 975 m/z Figure A21 UPLC MS mass spectra (bottom) and LC-IM-MS plot (top) of LC fraction #4 from the 1200 UP APG sample. 274