Desorption Electrospray Ionization Coupled with Ultraviolet Photodissociation for Characterization of Phospholipid Isomers in Tissue Sections

Transcription

1 Desorption Electrospray Ionization Coupled with Ultraviolet Photodissociation for Characterization of Phospholipid Isomers in Tissue Sections Dustin R. Klein, Clara L. Feider, Kyana Y. Garza, John Q. Lin, Livia S. Eberlin, and Jennifer S. Brodbelt Department of Chemistry, The University of Texas at Austin, Austin, TX Supporting Information Table of Contents Content Page no. 1. Experimental Methods S2-S3 2. Image of experimental setup and representative MS1 spectrum S4 3. HCD spectra, UVPD spectra and fragment ion maps for of m/z 760, m/z 782 and m/z HCD profiling spectra, UVPD profiling spectra and fragment ion maps for m/z HCD spectra, UVPD spectra and fragment ion maps for m/z 820 S9 6. Representative MS1 spectra from DESI profiling of human tissue S10 7. Representative HCD spectra from DESI profiling of human tissue S11 8. Representative UVPD spectra from DESI profiling of human tissue S12 9. HCD spectra, UVPD spectra and fragment ion maps for PC 18:1(6Z)/PC 18:1(6Z) and PC 18:1(9Z)/PC 18:1(9Z) 10. Representative expanded regions of UVPD mass spectra of varying concentration ratios of isomeric phospholipid standards S5-S7 11. DESI-UVPD ion imaging of a mouse brain tissue section S15-S DESI-UVPD ion imaging of a human brain tissue section S DESI-UVPD ion imaging of a lymph node tissue section with thyroid cancer metastasis 14. HCD and UVPD profiling spectra of regions cancerous and normal tissue from the lymph node tissue section with thyroid cancer metastasis 15. References S22 S8 S13 S14 S19 S20-S21 S1

2 Experiment Methods Materials Acetonitrile was purchased from EMD Millipore (Billerica, MA, USA). PC 18:1(9Z)/18:1(9Z) and PC 18:1(6Z)/18:1(6Z) standards were purchased from Avanti Polar Lipids (Alabaster, AB, USA). Hematoxylin stain was purchased from Ricca Chemical (Arlington, TX, USA) and eosin stain was purchased from Fisher Scientific (Waltham, MA, USA). Glass microscope slides and multi-spot glass microscope slides were purchased from Fisher Scientific (Waltham, MA, USA). Tissue Samples Mouse brain and kidney tissue samples were purchased from BioIVT (Hicksville, NY, USA). Pancreas, kidney, lung, fallopian and human tissues were acquired from Cooperative Human Tissue Network (CHTN). Ovarian tissue was acquired from MD Anderson Cancer Center Tissue Bank (Houston, TX, USA). Endometrial tissue was provided by Seton Medical Center (Austin, TX, USA). All tissues were analyzed in accordance with IRB and IBC protocols. Tissues were sectioned on a Cryostar NX50 cryostat (Thermo Scientific, San Jose, CA, USA) at 16 µm each and stored at -80 C prior to analysis. DESI-MS and DESI-UVPD Profiling and Imaging All experiments were performed in positive ion mode on an Thermo Scientific Orbitrap Fusion Lumos mass spectrometer (San Jose, CA, USA) modified with a 193 nm Coherent Excistar excimer laser (Santa Clara, CA, USA) to perform UVPD as previously described. 1 All spectra were collected with the instrument operating in full profile mode. DESI was performed using a 2D Omnispray DESI stage and ion source adapter (Prosolia, Inc., Indianapolis, IN, USA) with a custom manufactured ion transfer tube for coupling to the Fusion Lumos interface. Acetonitrile was sprayed at rate of 5-8 µl/min through a lab built sprayer using a nitrogen pressure of 180 psi and a spray voltage of 5kV. HCD spectra were collected at a normalized collision energy (NCE) of 30 and UVPD spectra were collected at 20 pulses with 6 mj per pulse. UVPD was performed in the high pressure trap of the dual linear ion trap with the q-value set to 0.1. For DESI profiling experiments, MS1, HCD and UVPD spectra were collected with a maximum ion injection time of ms and an AGC target of 1E5-1E6. MS1 profiling spectra were collected with a mass range of m/z and are presented with a mass range of m/z HCD and UVPD profiling spectra were collected with a mass range of m/z All representative profiling spectra are a composite of 15 consecutive averaged spectra and all representative spectra from S2

3 imaging data are a composite of 3 consecutive averaged spectra. DESI-MS images were collected at an instrument resolving power of 240,000 with the maximum ion injection time set to 50 ms and an ACG target of 1E6-2E6 to ensure consistent instrument scan times. DESI-UVPD images were collected by continuously activating the isolated m/z value of interest with the instrument operating at a resolving power of 120,000, the maximum ion injection time set to 1400 ms, the ACG target set to 1E6-2E6 and a mass range of m/z Mouse brain tissue section and human lymph node were imaged at a pixel size of 250 µm and human brain was imaged at a pixel size of 175 µm, respectively. 2D Imaging Data Analysis Thermo RAW files were converted to mzml files using msconvert (ProteoWizard). 2 Mzml files were subsequently imported into R using the mzr package from the Bioconductor repository. The peak intensity of an ion within a m/z window of.04 to.12 was taken. Ion intensities or ion ratio values were plotted as a heat map in R. Lipid Notation Lipid shorthand notation used is consistent with the notation previously described by Liebisch et al. 3 In brief, a / is used between acyl chains of known orientation with sn-1 followed by sn-2, and _ is used between acyl chains of unknown orientation. Double bond positions are indicated in parenthesis after the acyl chain composition as carbon number counted down the acyl chain starting at the carbonyl carbon followed by Z, E or Δ to indicate cis, trans or unknown double bond stereochemistry. S3

4 Figure S1. Picture of DESI stage set-up on an Orbitrap Fusion Lumos mass spectrometer and DESI-MS profiling mass spectrum of a mouse brain tissue section. The obvs The lipid profile observed in the positive ion mode MS1 spectra of a mouse brain tissue section was consistent with previous reports. 4 S4

5 Figure S2. HCD (NCE 30) mass spectra of ions of a) m/z 760, b) m/z 782 and c) m/z 798 from DESI profiling corresponding to the molecular ion, sodium-adducted ion, and potassium-adducted ion for PC 16:0_18:1, respectively. Fragment ion maps are shown in Supporting Information Figure S4. S5

6 Figure S3. UVPD (20 pulses, 6 mj) mass spectra of ions of a) m/z 760, b) m/z 782 and c) m/z 798 from DESI profiling corresponding to the molecular ion, sodium-adducted ion, and potassiumadducted ion or PC 16:0_18:1, respectively. Fragment ion maps are shown in Supporting Information Figure S4. S6

7 Figure S4. Fragment ion map for mass spectra in Supporting Information Figure 2 and Figure 3. Subscripts indicate the presence of a sodium (Na) or potassium (K) adduct. X represents either a proton, sodium cation or potassium cation. S7

8 Figure S5. a) HCD (NCE 30) and b) UVPD (20 pulses, 6 mj) mass spectra of the ion of m/z 826 obtained from DESI profiling of a mouse brain tissue section with corresponding fragment ion maps for detected isomers [PC 18:0_18:1(9 ), PC 18:0_18:1(11 )]. Subscripts indicate the presence of a potassium (K) adduct. S8

9 Figure S6. a) HCD (NCE 30) and b) UVPD (20 pulses, 6 mj) mass spectra of the ion of m/z 820 obtained from DESI profiling of a mouse brain tissue section with corresponding fragment ion map. Subscripts indicate the presence of a potassium (K) adduct. S9

10 Figure S7. MS1 spectra from DESI profiling of a) endometrial tissue, b) kidney tissue, c) lymph node tissue, d) ovarian tissue, e) pancreas tissue, and f) brain tissue. All tissues were human. S10

11 Figure S8. HCD (NCE 30) mass spectra from DESI profiling of a) endometrial tissue (m/z 782), b) kidney tissue (m/z 782), c) lymph node tissue (m/z 782), d) ovarian tissue (m/z 782), e) pancreas tissue (m/z 798), and f) brain tissue. All tissues were human. S11

12 Figure S9. UVPD (20 pulses, 6 mj) mass spectra from DESI profiling of a) endometrial tissue (m/z 782), b) kidney tissue (m/z 782), c) lymph node tissue (m/z 782), d) ovarian tissue (m/z 782), e) pancreas tissue (m/z 798), and f) brain tissue (m/z 798). All tissues were human. S12

13 Figure S10. a) HCD (NCE 30) and b) UVPD (20 pulses, 6 mj) mass spectra from DESI profiling of PC 18:1(6Z)/18:1(6Z). c) HCD (NCE 30) and d) UVPD (20 pulses, 6 mj) mass spectra from DESI profiling of PC 18:1(9Z)/18:1(9Z). Subscripts indicate the presence of a sodium (Na) adduct. S13

14 Figure S11. Representative expanded regions of UVPD (20 pulses, 6 mj) mass spectra of isomeric phospholipid standards, PC 18:1(6Z)/18:1(6Z) and PC 18:1(9Z)/18:1(9Z), in varying concentration ratios from DESI-UVPD ion imaging experiments. Mass spectra correspond to the following ratios of PC 18:1(6Z)/18:1(6Z) to PC 18:1(9Z)/18:1(9Z): a) 1:19, b) 1:9, c) 1:4, d) 1:1, e) 4:1, f) 9:1 and g) 19:1. S14

15 Figure S12. DESI-UVPD ion imaging of a mouse brain tissue section (replicate 1). a) Optical image of the H&E stained mouse brain tissue section, b) DESI-MS ion image of m/z 798, c) DESI- UVPD ion image of m/z 660, d) DESI-UVPD ion image of m/z 684, e) DESI-UVPD ion image of m/z 688, f) DESI-UVPD ion image of m/z 712, g) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ), h) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ) and i) DESI ratio image of the ratio of the summed intensities of the UVPD double bond diagnostic ions (I m/z )/(I m/z ). S15

16 Figure S13. DESI-UVPD ion imaging of a mouse brain tissue section (replicate 2). a) Optical image of the H&E stained mouse brain tissue section, b) DESI-MS ion image of m/z 798, c) DESI- UVPD ion image of m/z 660, d) DESI-UVPD ion image of m/z 684, e) DESI-UVPD ion image of m/z 688, f) DESI-UVPD ion image of m/z 712, g) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ), h) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ) and i) DESI ratio image of the ratio of the summed intensities of the UVPD double bond diagnostic ions (I m/z )/(I m/z ). S16

17 Figure S14. DESI-UVPD ion imaging of a mouse brain tissue section (replicate 3). a) Optical image of the H&E stained mouse brain tissue section, b) DESI-MS ion image of m/z 798, c) DESI- UVPD ion image of m/z 660, d) DESI-UVPD ion image of m/z 684, e) DESI-UVPD ion image of m/z 688, f) DESI-UVPD ion image of m/z 712, g) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ), h) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ) and i) DESI ratio image of the ratio of the summed intensities of the UVPD double bond diagnostic ions (I m/z )/(I m/z ). Images from this replicate are the ones used in the main text. S17

18 Figure S15. DESI-UVPD ion imaging of a human brain tissue section. a) Optical image of the H&E stained human brain tissue section, b) DESI-MS ion image of m/z 798, c) DESI-UVPD ion image of m/z 660, d) DESI-UVPD ion image of m/z 684, e) DESI-UVPD ion image of m/z 688, f) DESI-UVPD ion image of m/z 712, g) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ), h) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ) and i) DESI ratio image of the ratio of the summed intensities of the UVPD double bond diagnostic ions (I m/z )/(I m/z ). S18

19 Figure S16. DESI-UVPD ion imaging of a lymph node tissue section with thyroid cancer metastasis. a) Optical image of the H&E stained lymph node tissue section with thyroid cancer metastasis, b) DESI-MS ion image of m/z 798, c) DESI-UVPD ion image of m/z 660, d) DESI- UVPD ion image of m/z 684, e) DESI-UVPD ion image of m/z 688, f) DESI-UVPD ion image of m/z 712, g) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ), h) DESI-UVPD ion image of the summed intensity of m/z 660 and m/z 684 (I m/z ) and i) DESI ratio image of the ratio of the summed intensities of the UVPD double bond diagnostic ions (I m/z )/(I m/z ), j) DESI ratio image of m/z 684 to m/z 712. S19

20 Figure S17. HCD profiling spectra of regions of a) cancerous and b) normal tissue from the lymph node tissue section with thyroid cancer metastasis. S20

21 Figure S18. UVPD profiling spectra from regions of a) cancerous and b) normal tissue from the lymph node tissue section with thyroid cancer metastasis. Expanded UVPD spectra from regions of c) cancerous and d) normal tissue from the lymph node tissue section with thyroid cancer metastasis with further expanded insets. The DESI difference image for the lymph node tissue section with thyroid cancer metastasis was made from division of just m/z and m/z for avoid contributing signal form m/z S21

22 References (1) Klein, D. R.; Holden, D. D.; Brodbelt, J. S. Shotgun Analysis of Rough-Type Lipopolysaccharides Using Ultraviolet Photodissociation Mass Spectrometry. Anal. Chem. 2016, 88, (2) Kessner, D.; Chambers, M.; Burke, R.; Agus, D.; Mallick, P. ProteoWizard: Open Source Software for Rapid Proteomics Tools Development. Bioinformatics 2008, 24, (3) Liebisch, G.; Vizcaíno, J. A.; Köfeler, H.; Trötzmüller, M.; Griffiths, W. J.; Schmitz, G.; Spener, F.; Wakelam, M. J. O. Shorthand Notation for Lipid Structures Derived from Mass Spectrometry. J. Lipid Res. 2013, 54, (4) Škrášková, K.; Claude, E.; Jones, E. A.; Towers, M.; Ellis, S. R.; Heeren, R. M. A. Enhanced Capabilities for Imaging Gangliosides in Murine Brain with Matrix-Assisted Laser Desorption/Ionization and Desorption Electrospray Ionization Mass Spectrometry Coupled to Ion Mobility Separation. Methods 2016, 104, S22