Advantages of Ion Mobility Q-TOF for Characterization of Diverse Biological Molecules Add a New Dimension to your Research Capability with Agilent s New Drift Ion Mobility Q-TOF System
Overview: 6560 IM Q-TOF/MS System Design System Unmatched Resolution Addition Separation Power Enhanced Selectivity for Greater Sensitivity Various IM Q-TOF Applications Direct Collision Cross Section (CCS) Determination Summary 2
What Does Ion Mobility Bring to Mass Spectrometry? Adds Additional Separation Power A new dimension of separation for increased mass spectral purity especially for complex mixture analysis Improves Detection Limits Helps to eliminate interference from other analytes and background in the sample mixture Efficient ion focusing and transfer through the ion optics maximizes sensitivity for the overall system Enhances Compound Identification Improves confidence in compound identification and ion structure correlation through accurate collision cross section measurements Preserves Native Molecular Structure Effectively minimize ion heating effects to maintain native molecular conformations. 3
IM-QTOF Instrument Overview Based on 6550 Q-TOF Maintains QTOF performance Inserted trapping funnel and drift tube Trapping funnel & gate Matches IMS duty cycle with Q-TOF analysis Precursor ions are separated by drift time Next generation Ion Mobility QTOF 4
Basic Operational Principle of Ion Mobility: - For Conventional DC Uniform Field IMS V H Ion Mobility Cell V L Analyte Ions Detector Gating Optics Electric Field Stacked ring ion guide gives linear field Ω 5
Excellent in Resolution and Separation Power Chromatography Ion Mobility Mass ~seconds ~60 milli-seconds ~ 100 seconds 6
Ion Mobility Resolution: IM Resolution 530.7876 IM Resolution: 63.30 531.2889 MS Accuracy: < 1 ppm 531.7900 7
Ion Mobility Resolution - Continued Zipper Peptide: Resolution = 84! (Prof. David Russell, Texas A&M) 8
Separation of Isobaric Pesticides Theoretical Plot Aldicarb-sulfone (C 7 H 14 N 2 O 4 S) [M+Na] + = 245.056649 Acetamiprid (C 10 H 11 ClN 4 ) [M+Na] + = 245.056445 mass is 0.2 mda requires ~ 2,000,000 resolution! IMS Drift Separation 4 x10 4.5 4 19.441 3.5 3 2.5 2 1.5 1 0.5 0 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 6 x10 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Acetamiprid 18.297 Aldicarb-sulfone 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 Drift Time (ms) x10 4 +IMS DriftSpec (m/z: 245.013827-245.177238) (rt: 0.026-1.987 min) Aldicarbsulfone_A 5.5 * 18.297 5 4.5 4 3.5 * 19.441 3 2.5 2 1.5 1 0.5 0 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5 Drift Time (ms) 9
Separation of Isomers: Citrate and Isocitrate Isocitrate (C 6 H 8 O 7 ) Citrate (C 6 H 8 O 7 ) 10
Separation of Methylmalonic Acid (MMA) and Succinic Acid (SA): MMA (C 4 H 6 O 4 ) SA (C 4 H 6 O 4 ) MMA and SA are isomers which cannot be separated by traditional MS. MMA (Dimer) SA (Dimer) MMA & SA can be separated nicely by IMS. 11
Resolving Structural Sugar Isomers C 18 H 32 O 16 Raffinose Melezitose Resolving two isobaric tri-saccharides 12
Carbohydrates Analysis by IM-MS Lacto-N-di-fructose hexose I 60 Mixture of Lacto-N-di-fructose hexose I & II Fuc Fuc Gal 50 GlcNAc Gal Glc Ion Mobility Drift Time (ms) Lacto-N-di-fructose 40 hexose II Fuc Fuc 30 Gal GlcNAc Gal Glc 20 10 1018 1020 1022 1024 1026 1028 35 36 Lacto-N-difructose hexose II Mass (Da) 37 38 39 40Lacto-N-di- 41 42 fructose hexose I Drift Time (ms) 0 0 500 1000 1500 2000 Mass (Da) 35 36 37 38 39 40 41 42 Drift Time (ms) 13
Detecting Miss-formed Disulfide Bonds: Siamycin II 50 Mobility Drift Time (ms) 40 30 20 1085 1086 1087 1088 1089 1090 Mass (Da) 10 0 500 1000 1500 2000 Mass (Da) Siamycin II 30 31 32 33 34 35 36 37 38 39 40 41 Ion Mobility Drift Time (ms) 14
Ion Mobility Provides Greater Separation Protein Digest, Dr. Erin Baker, PNNL 15
Lipid Analysis: Mixture of L-α-phosphotidylethanolamine (PE) Lipids Ion Mobility Drift Time (ms) PE 19:N PE 48:N PE 46:N PE 44:N PE 40:N PE 38:N PE 37:N PE 36:N PE 34:N PE 33:N PE 29:N PE 63:N PE 61:N PE 59:N PE 92:N PE 90:N +1 lipids +2 lipids +3 lipids +4 lipids PE 38:8 PE 38:6 PE 38:4 PE 38:2 PE 38:0 PE 38:7 PE 38:5 PE 38:3 PE 38:1 PE 37:1 16
Ion Mobility Provides Greater Specificity 50 Mobility Drift Time (ms) 40 30 20 Integrated Mass Spectrum: 1444 1446 1448 1450 1452 1454 Mass (Da) Mobility-Filtered Mass Spectrum: 10 0 500 1000 1500 2000 Mass (Da) Crude bacterial extract (Prof. John McLean, Vanderbilt Univ.) 1444 1446 1448 1450 1452 1454 Mass (Da) S/N increased significantly! 17
Ion Mobility Provides Greater Specificity RNAseB Native Glycans Anaysis: Enable the extraction of ion series of interest - a group of glycans from matrix for further study. (Prof. Cathy Costello, Boston University) 18
Ion Mobility Simplifies Complex Spectra RNaseB Native Glycans (MS data before ion extraction) Glycan obscured by matrix can be identified Background noise greatly reduced! 19
Ion Mobility of Polymeric Ink Dispersants 20
Ion Mobility of Polymeric Ink Dispersants All Overlaid Extracted MS Spectra & Ion Mobility Heat Map Ion mobility can resolve overlapping charge-state isotopes unresolved by mass resolution 21
Ion Mobility of Diesel (Hydrocarbons) Sample Enable the extraction of ion series of interest for further study Background noise reduced significantly! 22
GC-APCI/IMS-QTOF analysis on ASTM compound mixture: RT: 4.3 min Some Possible Isomer Structures: 23
GC-APCI/IMS-QTOF analysis on ASTM compound mixture: RT: 3.4 min 1,2-Dimethylbenzene - Ortho-Xylene 1,3-Dimethylbenzene - Meta-Xylene 1,4-Dimethylbenzene - Para-Xylene Ethylbenzene 24
IMS-MS for Proteomics: Transmembrane Spanning Peptides of HeLa digest 30 25 All Ions Fragmentation Low dt 20 29 28 27 26 25 24 23 22 High dt Low dt 770 771 772 773 774 Drift Time (ms) vs. m/z 15 200 400 600 800 1000 1200 1400 1600 Drift Time (ms) vs. m/z High dt Low dt 25
Sensitivity - Detection limit of spiked compound in Urine Q-TOF mode IM mode 1 nm 10 nm 50 nm 100 nm 1000 nm 10000 nm 26
Ion Mobility Provides Greater Sensitivity! 6E+06 Area Response 600000 400000 200000 Linear Dynamic Range y = 59060x - 350.63 R² = 0.9993 Signal intensity (A.U.) 5E+06 4E+06 3E+06 2E+06 1E+06 0E+00 0 0 2 4 6 8 10 Sample Amount (pg) single pulse 1 ms trapping single pulse 4mstrapping multi-pulse 4x1 ms trapping single pulse 8 ms trap time multi-pulse 8x1 ms trapping Sensitivity: ~ 50 fg of Reserpine Dynamic range: ~ 3-4 orders Integrated signal intensity for tetrakis decyl ammonium bromide ion vs. ion trapping time for single pulse and multi-pulse experiments. Multiplexing experiments result in at least 10X higher signal intensity possibly due to less space charge effects and detector saturation issues. 27
IM Comparison on Cytochrom C (+8): (Uniform Drift Tube vs. Travelling Wave) S1: Native S2-S5: Denatured S1S3 Neutral Condition Denatured Condition Travelling Wave IM data RF: 90 V RF 90V RF 150V RF 90V RF 150V? S1 S2 S3 RF: 150 V S1 S2 Preserve protein native structures better! Due to the much lower ions heating. S4 S5 S4 S5 28
Ion Mobility Q-TOF on Protein Isoform Analysis Cry34AB 38 36 34 32 30 28 1346 1347 1348 1349 1350 Drift Time (ms) vs. m/z 29
Resolving Isoforms of IgG-2 mab Structure: (Publication Paul Schnier) Denaturated Condition: Only 1 conformation detected 30
IM Q-TOF Analysis of Native IgG-2: 27+ 27+ 26+ 25+ 28+ 28+ 24+ 30+ 29+ 29+ 23+ 23+ 22+ 21+ 21+ B A Two possible conformations detected 31
IM Q-TOF Analysis of Native IgG-1: 27+ 26+ 28+ 25+ 30+ 29+ 24+ 23+ 22+ 21+ B A Two possible conformations also detected 32
IM Q-TOF Comparison of IgG-1 and IgG-2: IgG-1 22+ IgG-1 (22+) A B IgG-2 22+ IgG-2 (22+) B A IgG-2 (22+ charge state) has more B form. 33
Determining Cross Sectional Areas Charge state of the analyte ion Charge on an electron Electric field Ω / /. Boltzmann constant Reduced mass of the ion and neutral Number density of the drift gas 0 273 760 Torr 2 Molecular size K 0 = Reduced Ion Mobility T = Temperature P = Pressure L = Drift length V = Voltage Drop across drift region t d = Drift time X-ray crystallography Ion mobility (using Helium) 34
Collision Cross Section Benchmark - Tetraalkylammonium Salts --- Vanderbilt University Analyte Measured Cross-Section [Å 2 ] TAA-4 166.61 ± 0.5% TAA-5 189.21 ± 0.6% TAA-6 212.71 ± 0.3% TAA-7 236.34 ± 0.2% TAA-8 257.19 ± 0.1% TAA-10 294.53 ± 0.1% TAA-12 323.62 ± 0.2% TAA-16 362.03 ± 0.2% TAA-18 381.58 ± 0.3% Literature Cross-Section [Å 2 ] 166.00 ± 0.3% 190.10 ± 0.1% 214.00 ± 0.3% 236.80 ± 0.2% 258.30 ± 0.4% Relative Standard Deviation [%] 0.56 0.28 0.41 0.01 0.24 High experimental precision (< 0.5% relative deviation) Agreement with literature (most < 0.5% deviation) 35
Cross Section Calculation of Ubiquitin Charge States m/z 612.8, [M+14H] 14+ m/z 659.8, [M+13H] 13+ Automated collision cross section calculation without the use of calibration curves approximate region for compact structures approximate region for elongated structures m/z 714.7, [M+12H] 12+ m/z 779.6, [M+11H] 11+ m/z 857.5, [M+10H] 10+ m/z 952.6, [M+9H] 9+ m/z 1071.6, [M+8H] 8+ m/z 1224.4, [M+7H] 7+ m/z 1428.4, [M+6H] 6+ m/z 1713.9, [M+5H] 5+ 1000 1500 2000 2500 3000 (Å) Reference: Koeniger and Clemmer J Am Soc Mass Spectrom 2007, 18, 322-331 36
Cross Section Calculation of Ubiquitin Charge States Ion Charge State CCS experimental (Å 2 ) [M+5H] 5+ 5 1196 [M+6H] 6+ 6 1431, 1658 CCS literature (Å 2 ) [M+7H] 7+ 7 1755, 1886 1910 [M+8H] 8+ 8 1966 1990 [M+9H] 9+ 9 2008 2090 [M+10H] 10+ 10 2114, 2197 2200 [M+11H] 11+ 11 2239, 2348 2340 [M+12H] 12+ 12 2412, 2511 2480 [M+13H] 13+ 13 2556, 2620 2600 [M+14H] 14+ 14 2680, 2726 Automated collision cross section calculation without the use of calibration curves Reference: Bush et al., Anal Chem. 2010, 82, 9557-9565. 37
Carbohydrates -- Great complexity by linkage Source: Blixt et al., PNAS, 2004 Current dominant strategies: MS n or Library searches 38
Conformational Space Occupancy of Biomolecules Hypothetical Ordering of Biomolecular Classes lipids peptides Collision Cross Section (Å 2 ) carbohydrates oligonucleotides Mass (Da) (Prof. John McLean, Vanderbilt U.) 39
A Word About Instrument Design LC Drift IMS MS and MS/MS High Resolution Accurate Mass Feature Drift Mobility TriWave Drift Mobility Advantage Mobility Resolution Highest (can be > 80) 80cm drift tube (L) higher voltage (E) No RF fields, Uniform low DC field Generally around 30 10cm drift TriWave, Multi-section device RF fields Over 2X the IM resolution of T-wave Sensitivity High efficiency ion funnels - trapping and rear Step wave lens Pressure barrier between Q and TriWave 10X to 50X better than T-wave Collision Cross Section (CCS) measurement (Ω) Direct determination of Ω Low electric field and constant drift tube pressure Ω cannot be directly determined from drift time. Need calibration tables. 1-2% precision Much better than T- wave (5-10%) Molecular structures Lower RF fields, less ion heating. Higher RF fields, tendency for higher fragmentation and ion heating Lower RF allows preservation of molecular structures LC Q IMS MS High Resolution Accurate Mass 40
Summary Next generation of IM Q-TOF Technology Added dimension of separation based on size, charge and molecular conformation Resolve and characterize the complex samples -- Increased peak capacity Preservation of molecular structures Direct determination collision cross sections 41