ABSTRACT KHODJANIYAZOVA, SITORA. Characterization of Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

ABSTRACT

KHODJANIYAZOVA, SITORA. Characterization of Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Mass Spectrometry Imaging (MSI) for Detection of Metabolites and Lipids in Pharmaceutical Pills, Soft and Hard Biological Tissues. (Under the direction of Dr. David C. Muddiman).

The principal advantages of mass spectrometry imaging (MSI) over conventional imaging systems used for evaluation of human health and disease (e.g., MRI, PET, X-ray, ultrasound, etc.) are the sensitivity, accuracy, and capability of detecting hundreds to thousands of molecules (both known and unknown) simultaneously. Even though there are a myriad of commercial and home built MSI systems available today, improvements are still necessary for MSI to have a significant impact on a diverse range of applications. The work described here details efforts towards the development of an innovative MSI platform named infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). IR-MALDESI is an ambient ionization source that uses a mid-IR laser to desorb neutral species from a sample followed by ionization in an orthogonal electrospray beam. IR-MALDESI is an innovative MSI techniques in both targeted and untargeted analyses. This is due to its unique combination of attributes offering complementary information to other MSI techniques including: minimal sample preparation, operation at atmospheric pressure, quantitative ablation and sampling which enables relative and absolute quantification, 3- dimensional (3D) capabilities, and soft ionization of a broad range of compound classes without chemical derivatization. This research thesis describes the following characteristics of IR-

MALDESI platform: spectral accuracy, lipidome coverage from fresh and formalin-fixed paraffin- embedded (FFPE) tissues, feasibility of IR-MALDESI for 3D MSI and direct analysis of undecalcified bones.

IR-MALDESI experiments rely heavily on MS1 data since each voxel is fully ablated after a few laser shots making MS/MS on multiple features from the same voxel very challenging.

Knowing that accurately measured mass-to-charge (m/z) ratios and relative ion abundances within an isotopic envelope aid in revealing elemental compositions, we investigated how the Orbitrap ion count affects mass and spectral accuracy. Our findings were implemented in untargeted MSI analyses of healthy and disease-affected tissues to confirm the presence of one or more analytes of interest using sulfur, carbon, and nitrogen counting.

In untargeted IR-MALDESI analyses of biological samples, a significant number of abundant peaks corresponding to lipids are observed. However, the fraction of the lipidome that can be extracted and ionized by IR-MALDESI has not been defined. Our findings show that IR-

MALDESI detects 70 unique lipid ions that are not observed in more generic analyses such as direct infusion (also known as shotgun).

Histology laboratories around the world fix fresh tissues in formalin to preserve cellular morphology that can help with diagnosis and prognosis of a disease. However, fixation denatures biomolecules causing challenges for non-optical methods of detection such as MSI. Our results show that IR-MADLESI is compatible with direct analysis of FFPE tissues and that abundance of all common ions detected from both fresh and FFPE tissue is comparable.

To uncover additional spatial information that cannot be collected using traditional planar

MSI, an over-the-counter (OTC) pharmaceutical pill was used to test the utility of IR-MALDESI for 3D MSI. Without consecutive cryosectioning, 3D IR-MALDESI MSI was performed by repeatedly acquiring 2D images over the same region of interest. We investigated depth resolution as a function of laser energy and achieved 2.3 μm depth resolution with reduced laser power.

The gold standard for preparation of bone tissue prior to MSI is decalcification, where fresh bone is soaked in various solvents days, causing chemical and physical changes. This process removes minerals, making it easier to cut into thin slices. Only two MSI sources are reported for the analyses of bone tissues: MALDI and SIMS, both requiring decalcification. Using IR-

MALDESI source, an MSI method was developed for analysis on fresh, unmodified bones.

Healthy and stroke-affected mouse bones were imaged resulting in 826 and 669 putatively annotated features.

Characterization of Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR- MALDESI) Mass Spectrometry Imaging (MSI) for Detection of Metabolites and Lipids in Pharmaceutical Pills, Soft and Hard Biological Tissues

by Sitora Khodjaniyazova

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Chemistry

Raleigh, North Carolina 2021

APPROVED BY:

______David C. Muddiman Edmond F. Bowden Chair of Advisory Committee

______Gavin J. Williams H. Troy Ghashghaei

DEDICATION

To my parents, Tatyana and Shavkat.

BIOGRAPHY

Sitora Khodjaniyazova was born to her parents Tatyana and Shavkat on May 2, 1993 in

Tashkent, Uzbekistan. In middle school Sitora started failing introductory Chemistry class.

Fortunately, her dad had time and energy to spend long evenings on weekdays and weekends solving chemistry problems from textbooks, eventually helping Sitora get much better in

Chemistry. In high school, it was clear to Sitora that Chemistry, with a combination of

Mathematics, was the only class she was truly passionate about. In 2012 she moved to Buffalo,

New York to pursue a Bachelor of Science degree in Chemistry with a minor in Mathematics at

SUNY Buffalo. During her time in Buffalo, she also engaged in undergraduate research for

Professor Frank V. Bright, under the direct supervision of Sidney G. Coombs (a senior graduate student at the time). In the Bright Group, Sitora investigated chemistry on the surface of chemically modified porous silicon wafers using photoluminescence (PL) and Fourier transform-infrared (FT-

IR) spectroscopy imaging. Undergraduate research was a pivotal point in Sitora’s life because that is exactly when she realized that research in analytical chemistry is something she would really enjoy doing after graduation. This realization led to the next steppingstone in her career: in 2016

Sitora moved to Raleigh, North Carolina to pursue a Ph.D. in analytical chemistry under the supervision of Professor David C. Muddiman. In the Muddiman Group Sitora was introduced to mass spectrometry which has been her passion ever since. In the Muddiman Group, Sitora flourished not only as an analytical chemist and mass spectrometrist but also as a person, thanks to wonderful co-workers, mentors, and an advisor, with which she had the privilege of working.

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ACKNOWLEDGMENTS

I would like to thank my parents and brother for unconditional love, support, and patience.

I love you and cannot thank you enough. My parents helped me and my brother to immigrate to the United States where I met three key figures in my career: Bonnie L. Bright (undergraduate academic advisor), Professor Frank V. Bright (undergraduate research advisor), and Professor

David C. Muddiman (Ph.D. advisor). Bonnie, Frank, and Dave helped me to become a scientist I am today. I thank you all.

Bonnie L. Bright was the first person at SUNY Buffalo who convinced me that research in analytical chemistry might be something I would really enjoy, and she was right. I cannot thank

Bonnie enough. Bonnie saw something in me that I could not see in myself. In spring of 2014,

Bonnie encouraged me to reach out to her husband, Frank V. Bright, who was a SUNY

Distinguished professor of Chemistry at SUNY Buffalo at the time. I ended up working in Frank’s laboratory under the direct supervision of Dr. Sidney G. Coombs, a senior graduate student at the time. I would like to thank Dr. Coombs for all the hard work and patience during my time in the

Bright Group: she was an amazing mentor and teacher. Thanks to Sidney, I succeeded as an undergraduate researcher: I presented my work at multiple different conferences, and most importantly I learned how to work independently in a chemistry laboratory as well as analyze and interpret data. I would also like to thank former Bright Group students (Dr. Dustin T. McCall, Dr.

Joel F. Destino, Dr. Ian J. Horner, Dr. Crystal M. Collado, Dr. Samantha Matthews, Jennifer M.

Empey, and David Szczur) and postdoctoral researcher at the time, Dr. Shruti Trivedi, for their support, constructive criticism, and sense of humor. And, again, I would like to thank Professor

Frank V. Bright for his guidance, especially during my senior year of college when I was making decisions about graduate schools. Frank convinced me that I can succeed in graduate school. He

also made me believe that I can achieve anything I want if I work hard enough and with the right advisor who can teach and guide me during the next 4-6 years of graduate school. Frank was right, because later in life I met Professor Muddiman, my current Ph.D. advisor, who is the only person in my life who helped me tremendously with both professional and personal growth.

Professor Muddiman is an extraordinary scientist, teacher, leader, and role model who shaped me not only as an analytical chemist but also as a person. And I cannot thank him enough for this. I first met Professor Muddiman in March 2016 during the visiting weekend at North

Carolina State University (NCSU) and, to my surprise, I knew right away I wanted to work in his laboratory. His enthusiasm and excitement about science were and still are contagious. In June

2016, I joined his group. Since the summer of 2016, there was not a single day when I felt alone or unsupported in graduate school because Professor Muddiman always makes time for his students no matter what. He taught me how think critically and most importantly independently.

He never told me what to do in the chemistry laboratory. Instead, he would ask me questions so that I could come up with my own solutions. Also, Professor Muddiman showed me that life is all about attitude: no matter how bad things are, there is always a way out. I am extremely lucky to get to work with Professor Muddiman over the past 4.5 years. Professor Muddiman will be forever my mentor and friend, and I cannot thank him enough for tremendous support and guidance with everything during my time in graduate school.

I am proud to be part of the Muddiman Group because people in the group are talented, genuine, ambitious, and hardworking. I would like to thank current members of the Muddiman

Group for always being enthusiastic team-players and wonderful co-workers: Kenneth P. Garrard,

Jaclyn G. Kalmar, Anqi Tu, M. Caleb Bagley, Crystal L. Pace, Hongxia (Hellena) Bai, Allyson L.

Mellinger, Theresse M. Robinson, and Kaylie I. Kirkwood. I would not be able to accomplish

much in graduate school without your help. Every one of you taught me something that I find valuable inside and outside the chemistry laboratory. I would also like to thank former graduate students who spent their time and energy training me: Dr. Milad Nazari and Dr. Måns Ekelöf.

Milad, thank you for everything you did for me during my first 1.5 years in graduate school: training me, answering all my questions about science, teaching how to use different instruments and write papers. Måns, thank you for always finding the time to talk about science. You have been a source of enormous support during the final years of my graduate studies. I would also like to thank Dr. Mark T. Bokhart, Dr. James P. McCord, Dr. Elizabeth S. Hecht, Dr. Philip L. Loziuk, and Samuel R. King for constructive criticism during group meetings. Finally, I would like to thank all collaborators inside and outside the Chemistry department because without you none of the science would be possible.

Last, but not the least, I want to thank my significant other, Tyler Kropiewnicki, for his unconditional support with everything in my life. I love you very much.

TABLE OF CONTENTS

LIST OF TABLES ...... x LIST OF FIGURES ...... xi CHAPTER 1: Fundamentals of Mass Spectrometry for Applications in Imaging ...... 1 1.1 Basic Principles of Mass Spectrometry ...... 1 1.1.1 A Brief Overview of Mass Spectrometry ...... 1 1.1.2 The Origins of Mass Spectrometry ...... 1 1.1.3 Relation of Mass and Energy ...... 2 1.1.4 Isotopic Distributions: The Role of Isotopes in Mass Spectrometry...... 3 1.1.5 Isotopic Distributions Can Help Predict Elemental Compositions ...... 4 1.2 How ESI and MALDI Revolutionize Biological Mass Spectrometry ...... 5 1.2.1 ESI (Electrospray Ionization) ...... 5 1.2.2 MALDI (Matrix-Assisted Laser Desorption Ionization) ...... 7 1.3 IR-MALDESI - one of the Softest Ionization Sources ...... 8 1.4 IR-MALDESI Source Coupled with a Q Exactive Plus Mass Spectrometer ...... 9 1.5 IR-MALDESI Mass Spectrometry Imaging ...... 11 1.6 References ...... 12

CHAPTER 2: Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer using Isotope Ratio Mass Spectrometry ...... 16 2.1 Introduction ...... 16 2.2 Experimental Section ...... 18 2.2.1 Materials ...... 18 2.2.2 Direct Infusion of Caffeine, MRFA, and Ultramark Mixture Using Q Exactive Plus 19 2.2.3 Direct Infusion of IRMS Reference Standards Using Q Exactive Plus ...... 19 2.2.4 Analysis of CaffeineSigma, MRFA, and Ultramark Using IRMS ...... 20 2.2.5 Data Analysis ...... 21 2.3 Results and Discussion ...... 22 2.3.1 Orbitrap’s Sensitivity of Measuring Relative Abundances across Eight AGC Targets...... 22 2.3.2 Orbitrap’s Sensitivity of Carbon Counting across Eight AGC Targets ...... 28 2.4 Conclusions ...... 39 2.5 Acknowledgments ...... 40 2.6 References ...... 41

CHAPTER 3: Infrared Matrix-Assisted Laser Desorption Electrospray Ionization and Shotgun Provide Complementary Information about Lipidome Coverage ...... 44 3.1 Introduction ...... 44 3.2 Methods ...... 47 3.2.1 Materials ...... 47 3.2.2 Preparation of Rat Liver for Shotgun and IR-MALDESI MSI ...... 48 3.2.3 Direct Infusion ...... 48 3.2.4 IR-MALDESI MSI ...... 49 3.2.5 Data Collection and Analysis ...... 49 3.3 Results and Discussion ...... 51

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3.3.1 Lipidome Coverage via IR-MALDESI and Shotgun ...... 51 3.4 Conclusions ...... 60 3.5 Acknowledgements ...... 61 3.6 References ...... 62

CHAPTER 4: Direct Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) and Fresh Flash-Frozen Tissues using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Mass Spectrometry Imaging 68 4.1 Introduction ...... 68 4.2 Methods ...... 70 4.2.1 Materials ...... 70 4.2.2 Rat Liver ...... 70 4.2.3 Flash-freezing Protocol ...... 70 4.2.4 Preparation of FFPE Tissue ...... 71 4.2.5 IR-MALDESI MSI ...... 72 4.3 Results and Discussion ...... 73 4.4 Conclusions ...... 80 4.5 Acknowledgements ...... 80 4.6 References ...... 81

CHAPTER 5: Three-Dimensional Imaging with Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry ...... 84 5.1 Introduction ...... 84 5.2 Experimental Section ...... 86 5.2.1 Materials ...... 86 5.2.2 IR-MALDESI System ...... 87 5.2.3 Depth Resolution Determination ...... 88 5.2.4 2D MSI ...... 88 5.2.5 3D MSI ...... 88 5.2.6 Data Processing ...... 88 5.3 Results and Discussion ...... 89 5.3.1 Depth Resolution Determination on a Pill ...... 89 5.3.2 2D MSI on a Pill Microtomed in Half ...... 89 5.3.3 3D MSI on the Pill Microtomed in Half ...... 93 5.4 Conclusions ...... 96 5.5 Acknowledgments ...... 96 5.6 References ...... 97

CHAPTER 6: Mass Spectrometry Imaging (MSI) of Fresh Bones using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) ...... 100 6.1 Introduction ...... 100 6.2 Experimental ...... 103 6.2.1 Mouse Bones ...... 103 6.2.2 Flash-freezing ...... 103 6.2.3 Cryosectioning ...... 104 6.2.4 IR-MALDESI MSI ...... 105 6.2.5 Optical Images ...... 106

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6.2.6 Data Analysis ...... 106 6.2.7 Embedding Materials other than Plaster of Paris ...... 107 6.2.8 Data Availability ...... 108 6.3 Results and Discussion ...... 108 6.3.1 Laser Focus in IR-MALDESI Imaging...... 108 6.3.2 Challenges of Bone Imaging using IR-MALDESI ...... 109 6.3.3 Embedding Protocol for IR-MALDESI MSI of Mouse Bones ...... 110 6.3.4 Criteria for Embedding Material ...... 112 6.4 Conclusions ...... 117 6.5 Acknowledgements ...... 118 6.6 References ...... 119

APPENDICES ...... 122 Appendix A: Supplemental Information for Chapter 2 ...... 123 Figure A.1 ...... 123 Figure A.2 ...... 124 Figure A.3 ...... 125 Figure A.4 ...... 126 Figure A.5 ...... 126 Figure A.6 ...... 127 Figure A.7 ...... 127 Appendix B: Supplemental Information for Chapter 3...... 128 Figure B.1 ...... 128 Figure B.2 ...... 129 Table B.1 ...... 130 Table B.2 ...... 140 Table B.3A ...... 142 Table B.3B ...... 151 Table B.3C ...... 159 Table B.4A ...... 160 Table B.4B ...... 200 Appendix C: Supplemental Information for Chapter 4 ...... 316 Table C.1 ...... 316 Table C.2 ...... 319 Table C.3 ...... 319 Figure C.1 ...... 321 Appendix D: Supplemental Information for Chapter 5 ...... 322 Table D.1...... 322 Table D.2...... 322 Figure D.1 ...... 323 Figure D.2 ...... 324 Figure D.3 ...... 325 Appendix E: Supplemental Information for Chapter 6...... 326 Figure E.1 ...... 326 Table E.1 ...... 326 Table E.2 ...... 336

LIST OF TABLES

Table 2.1 Analysis of Four IRMS Reference Standards Using a Q Exactive Plus ...... 27 Table 2.2 Comparison of Absolute Monoisotopic Ion Abundances Acquired at Small (column 2) and Large (column 3) Total Ion Populations ...... 33 Table 6.1 List of embedding materials used for IR MALDESI MSI of mouse bones ...... 113

LIST OF FIGURES

Figure 1.1 Representative isotopic distribution ...... 3 Figure 1.2 Schematic of ESI (in positive mode). ESI voltage can be either positive or negative depending on the physiochemical properties of an analyte ...... 7 Figure 1.3 Schematic of IR-MALDESI ...... 9 Figure 1.4 Schematic of Q Exactive Plus coupled to IR MALDESI Image courtesy of Thermo Fisher Scientific ...... 10 13 13 Figure 2.1 Dependence of calculated δOrbitrap C on absolute ion abundance of A+1 ( C1) peak using caffeineSigma, MRFA, and ultramark 1421 ...... 25 Figure 2.2 Orbitrap mass spectra collected in positive ESI mode at RPfwhm of 140 000 at m/z 200 for glutamic acid 1 (USGS-40), caffeineIAEA (IAEA-600), sulfanilamide, and glutamic acid 2 (USGS-41) that were injected separately one sample at a time ...... 26 Figure 2.3 Representative Orbitrap mass spectra for caffeineSigma, MRFA, and ultramark 1421 collected in positive ESI mode at RPfwhm of 140 000 at m/z 200 and AGC targets of 5 × 105, 3 × 106, and 2 × 105, respectively ...... 29 Figure 2.4 Deviations from the known number of carbons across different ion populations in caffeineSigma, MRFA, and ultramark 1421...... 32 2 Figure 2.5 Pearson χ distributions for caffeineSigma, MRFA, and ultramark 1421 across eight AGC targets ...... 36 Figure 2.6 Proposed workflow for elucidation of unknown elemental compositions in untargeted MSI experiments ...... 38 Figure 3.1 MS2 and MS1 for FA (20:4) detected in shotgun and IR-MALDESI analyses ...... 53 Figure 3.2 Representative MS1 spectra collected by shotgun and IR-MALDESI ...... 54 Figure 3.3 Venn diagram showing the number of lipid ions, lipid IDs, and lipid classes detected via IR-MALDESI and shotgun analyses ...... 56 Figure 3.4 Abundance ratios and MMA distributions for lipid ions detected in shotgun and IR-MALDESI ...... 57 Figure 3.5 The number of unique lipid IDs that were annotated only via shotgun or only via IR-MALDESI ...... 59 Figure 3.6 Venn diagram: the number of lipid ions that can be recovered from shotgun and IR-MALDESI analyses ...... 60 Figure 4.1 Flash freezing protocol ...... 71 Figure 4.2 Experimental design ...... 73 Figure 4.3 Representative MS1 spectra collected from formalin-fixed paraffin-embedded (FFPE) and flash-frozen (FF) rat liver tissues ...... 75 Figure 4.4 Abundance of 823 tissue-specific precursor ions that were detected both in FF and FFPE tissues ...... 76 Figure 4.5 Venn diagram showing the number of lipid ions, IDs, and classes that were annotated by LipidSearch ...... 78 Figure 4.6 The number of lipid ions that can be recovered from FF and FFPE tissues ...... 78 Figure 4.7 Abundances of 44 common lipid ions that were identified using MS2 data from either one or both FF and FFPE tissues ...... 79 Figure 5.1 Pills used as a model for demonstration ...... 87

Figure 5.2 Ablation depth, optical, and laser images were obtained using a confocal laser scanning microscope after ablation of 1, 5, 10, 15, and 20 layers ...... 90 Figure 5.3 Representative mass spectra for omeprazole (C17H19N3O3S), triethyl citrate (C12H20O7), and monomers of starch (C6H10O5) ...... 91 Figure 5.4 Distribution of pill-specific molecules in a pill trimmed in half (2D images) ...... 92 Figure 5.5 Distribution for A peaks of starch, triethyl citrate, and omeprazole on the half pill across 50 layers with laser spot size 80 μm and depth resolution 16.3 μm ...... 94 Figure 5.6 3D intensity maps for starch, triethyl citrate, and omeprazole ...... 95 Figure 6.1 Optical image of a 50 μm-thick mouse tibia embedded in carboxymethyl cellulo se (CMC)/gelatin medium ...... 107 Figure 6.2 Optical image of mouse tibia cut in half ...... 107 Figure 6.3 Schematic illustrating how half of a bone would be positioned on the IR-MALDESI stage and under the IR laser ...... 110 Figure 6.4 Proposed protocol for bone embedding ...... 111 Figure 6.5 IR-MALDESI MSI of a stroke-affected mouse humerus embedded in Plaster of Paris (optical images, heatmap, mass spectra) ...... 114 Figure 6.6 IR-MALDESI MSI of healthy and stroke-affected mouse humeri embedded in Plaster of Paris (optical image and heatmap) ...... 116

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CHAPTER 1

Fundamentals of Mass Spectrometry for Applications in Imaging

1.1 Basic Principles of Mass Spectrometry

1.1.1 A Brief Overview of Mass Spectrometry

Mass spectrometry is an analytical technique used for measurements of mass-to-charge

(m/z) ratios and relative abundances of ionized species (e.g., atoms, molecules, clusters which can be atoms and/or molecules held together by a binding force). A typical mass spectrometer consists of an ion source (where neutral species are transformed into gas-phase ions), m/z filter (where ions with specified m/z ratios are allowed to pass deeper into a mass spectrometer), and finally mass analyzer (where m/z ratios and abundances are measured in an ultra-high vacuum). Charged species respond differently to some combination of magnetic and/or electric fields applied to them, and by measuring changes in ion trajectories due to those fields, the mass of an individual ion can be determined.

1.1.2 The Origins of Mass Spectrometry

A lot of people contributed to the field of mass spectrometry. For example, in 1815 the

British physician W. Prout formulated theory about multiple hydrogens which states that molecular masses are multiples of the mass of hydrogen. His theory played an important role in the discussion about isotopes (elements with different number of neutrons but the same number of protons and electrons). In 1869 the Russian chemist D. Mendeleev for the first time presented his periodic table that illustrated chemical and physical properties of some elements. In 1897 J. J. Thomson

(physicist) discovered the electron, and in 1911 E. Rutherford (physicist as well) discovered atomic nucleus. In 1914 T. W. Richards was able to show that mass of an ordinary lead was different from

1 the mass of lead found in radioactive minerals. Significant mass difference between the two provided evidence for the theory of isotopes.1

1.1.3 Relation of Mass and Energy

Discovery of isotopes allowed for determination of elemental compositions with far greater accuracy than before. Isotopes are atoms that have the same number of protons and electrons but different number of neutrons (e.g., 13C and 12C). Isotopologues are molecules that have different

13 16 12 18 isotopic compositions with one or more substituted isotope. For example, C O2, C O2,

13 18 12 16 C O2 are isotopologues of C O2. The word “isotope” comes from two Greek roots “isos” and

“topos” meaning “the same place”, because different isotopes of the same element occupy the same position on the periodic table. For example, both 12C and 13C represent carbon atom on the periodic table, even though 13C has one extra neutron compared to 12C. Mass difference between

13C and 12C is 1.0034 Daltons (Da), which is not the mass of a neutron. If the same manipulations are done with two nitrogen isotopes (15N and 14N), the mass difference turns out to be 0.9970 Da, which is also not the mass of a neutron. This phenomenon can be explained by Einstein's nuclear binding energy (ΔE) equation which describes the amount of energy that must be supplied to a nucleus in order to move neutrons and protons away from each other:

binding energy = ΔE = Δm × c2 (Equation 1.1)

Δm = mass defect= [(# p × mass of p) + (# n × mass of n)] – mass of nucleus

p = proton, n = neutron

c = speed of light = 3.0 × 108 m/s

Just as electrons orbiting the nucleus have preferred configurations, each nucleon has its own unique binding energy as well. It worth noting that electrons also have binding energies, called

2 ionization potential, however, ionization potential of a single electron is approximately a million times smaller than the binding energy of a single proton or neutron in a nucleus.

1.1.4 Isotopic Distributions: The Role of Isotopes in Mass Spectrometry

Due to the differences in binding energies, we can identify what elements make up an unknown by monitoring mass shifts between adjacent peaks in isotopic distributions. Isotopic distribution shows masses and abundances of a monoisotopic molecule and its isotopologues

(Figure 1.1). The monoisotopic peak in isotopic distribution shows mass and abundance of a molecule composed of the most abundant isotopes (e.g., 12C, 14N, 1H, 16O, etc.). Peaks to the right of the monoisotopic peak represent isotopologues. To determine what isotope corresponds to the

Figure 1.1. Representative isotopic distribution. second peak in isotopic distribution, we need to find the mass difference between monoisotopic peak and the first isotopologue. In this example (Figure 1.1), the m/z difference is 1.0034 Da which corresponds to the mass difference between 13C and 12C.2 Therefore, the second peak in isotopic distribution represents a molecular ion with one 13C and N-1 12C isotopes (N being the total number of carbons).

1.1.5 Isotopic Distributions Can Help Predict Elemental Compositions

From isotopic distribution we can also determine the total number of carbons in the unknown because the relative abundance of 13C isotopologue (or height of 13C peak in isotopic distribution) changes with the total number of carbons in a molecule. This has to do with the number of permutations or arrangements with regard to the order of one 13C in a molecule: the number of permutations increases with the total number of carbons. For example, one 13C has 2 permutations in ethane:

13 12 CH3 CH3

12 13 CH3 CH3

Similarly, one 13C has 4 permutations in butane:

13 12 12 12 CH3 CH3 CH3 CH3

12 13 12 12 CH3 CH3 CH3 CH3

12 12 13 12 CH3 CH3 CH3 CH3

12 12 12 13 CH3 CH3 CH3 CH3

Therefore, the relative abundance of 13C isotopologue in a molecule with 4 carbons will always be higher than the relative abundance of 13C isotopologue in a molecule with 2 carbons.

The number of carbons can be estimated by dividing the relative abundance of 13C isotopologue

(e.g., 8.8% from Figure 1) by the relative abundance of one 13C isotope in nature:2

푟푒푙푎푡𝑖푣푒 푎푏푢푛푑푎푛푐푒 표푓 𝑖푠표푡표푝표푙표푔푢푒 푤𝑖푡ℎ 표푛푒 13퐶 8.8% 푒푠푡푖푚푎푡푒푑 푛표. 표푓 푐푎푟푏표푛푠 = = = 8 (Equation 1.2) 푟푒푙푎푡𝑖푣푒 푎푏푢푛푑푎푛푐푒 표푓 표푛푒 13퐶 𝑖푠표푡표푝푒 𝑖푛 푛푎푡푢푟푒 1.1%

If isotopologues of other elements are resolved in isotopic distribution, Equation 1.3 can be used to estimate the number of any other element:

푟푒푙푎푡𝑖푣푒 푎푏푢푛푑푎푛푐푒 표푓 𝑖푠표푡표푝표푙표푔푢푒 푤𝑖푡ℎ 표푛푒 푋 𝑖푠표푡표푝푒 푒푠푡푖푚푎푡푒푑 푛표. 표푓 푎푡표푚푠 = (Equation. 1.3) 푟푒푙푎푡𝑖푣푒 푎푏푢푛푑푎푛푐푒 표푓 표푛푒 푋 𝑖푠표푡표푝푒 𝑖푛 푛푎푡푢푟푒

1.2 How ESI and MALDI Revolutionized Biological Mass Spectrometry

There is a myriad of different ionization sources that range from hard to soft. Harder ionizations result in excessive fragmentation patterns of a precursor ion (e.g., precursor ion ABC+ dissociates into its component ions A+, B+, C+); while in softer ionizations, charge is deposited onto a precursor molecule itself (e.g., ABC+ or ABC+adduct ion). The work described in this thesis is focused only IR-MALDESI, one of the softest ionization sources available today, but before talking about this source in more detail, ESI and MALDI will be introduced first because IR-

MALDESI is a hybrid of ESI and MALDI.

1.2.1 ESI (Electrospray Ionization)

When choosing between ionization methods, one should consider the physical state of an analyte because some ionization techniques require the analyte to have certain physical properties.

For example, electron ionization (EI) is limited to small, initially neutral, thermally stable, and volatile compounds because neutral sample must be “boiled off” and converted into a gas phase prior to bombardment with an electron beam. Thermally unstable compounds can decompose in the heating chamber prior to interaction with the electrons. EI is considered a hard ionization source because energy delivered in the system from the electron beam is high enough to break multiple bonds in a molecule. Fragmentation of precursor ion produces a very detailed

“fingerprint” that can be searched against a database to obtain structural information.

Until not too long ago, neutral molecules could be ionized only via evaporation and interaction with an electron or photon beam. However, the problem with this classic method of ion production is that it requires the species of interest to be in vapor phase as individual free molecules. And when it comes down to analysis of biological samples, we know that thermal stress catastrophically decomposes biological molecules. The big question was how to make ions out of

5 molecules that cannot vaporize.

In 1984, building on ideas of Dole3-5, Iribarne and Thomson6, Professor John B. Fenn developed an electrospray ionization (ESI) source for analyses of macromolecules.7,8 In 2002 he received one-quarter of the Nobel Prize in Chemistry due to the tremendous impact of ESI on the field of bioanalytical chemistry.9 For the first time, a charge could be deposited on intact, unfragmented biomolecules (e.g. proteins) which were later described as “flying elephants” because these biological ions were very large compared to typical gas-phase ions.

ESI is the softest ionization technique, which allows for analysis of non-covalent complexes, polar, neutral, big, and small molecules at ambient atmosphere. A typical ESI source consists of a capillary and emitter tip connected with a metallic junction, an MS inlet, and voltage difference between the ESI emitter tip and MS inlet (Figure 1.2). To generate ions in the source, potential (2-5 kV) is applied between capillary and emitter tip in liquid junction through which water-based, conductive solution with analyte is flowing. As a result, oxidation or reduction of water generates protons or hydroxides that are evenly distributed on the surface of each droplet:

+ - 2H2O → O2 + 4H + 4e (dominant reaction in positive ESI mode).

- - 2H2O + 2e → H2 + 2OH (dominant reaction in negative ESI mode).

When too many charges are accumulated at the emitter tip, a Taylor cone is formed as a result of pressure build-up. Electrostatic and hydrodynamic forces result in the formation of charged droplets as the solution is emitted from the capillary’s tip. Since ESI mimics an electric circuit, all charged droplets are drawn towards a very hot (~250°C) counter electrode (or ion transfer capillary) where excess of solvent is evaporated due to the collision of charged species with themselves and with ambient ions/molecules. Eventually, too many charges are present on the surface of a droplet. This is when the Rayleigh limit is reached: the point at which Coulombic

6 repulsion forces equal the surface tension of a droplet. As a result, Coulombic fission takes place several times, and offspring droplets (or progenies) with charges on them are born.

There are two models that describe how gas-phase ions are formed in ESI. The first one is the charge-residue model proposed by M. Dole: offspring droplet will keep evaporating and undergoing fission until only one analyte ion is left in the droplet.3,10 Residual solvent then evaporates in MS inlet and charge(s) is (are) retained on the analyte molecule. The second model is the ion evaporation model proposed by J. V. Iribarne and B. A. Thomson; this is the model that

John B. Fenn believed in.6,10 The model states that Coulombic fission and solvent evaporation will continue, until the droplet is so small and charge density is so high that electrostatic field at the droplet’s surface “lifts” analyte ions into the gas.

Figure 1.2. Schematic of ESI (in positive mode). ESI voltage can be either positive or negative depending on the physiochemical properties of an analyte.

1.2.2 MALDI (Matrix-Assisted Laser Desorption Ionization)

Another soft ionization method that revolutionized biological mass spectrometry is matrix- assisted laser desorption ionization (MALDI). At the same time as J. B. Fenn, K. Tanaka received one-quarter of the 2002 Nobel Prize in Chemistry for demonstrating that proteins can be ionized when proper matrix and laser wavelength are used.11 In MALDI, analyte compounds are embedded

7 in a matrix, consisting of small organic molecules. Upon laser excitation, analyte and matrix co- desorb from the surface and multiple charge-transfer reactions occur between matrix-matrix, matrix-analyte, and analyte-analyte. Residual energy drives matrix-analyte separation and charge transfer. Unlike ESI, MALDI can ionize samples with high salt concentrations. Also, MALDI produces primarily singly charged species making spectra interpretation easier. The reason for this is currently unknown; however, there are two proposed theories for ionization mechanisms in

MALDI: gas-phase protonation and lucky survivor theory.12 Gas-phase protonation theory states that matrix molecules are ionized first upon irradiation followed by proton transfer between matrix and analyte molecules in gas phase.12 Lucky survivor theory predicts that excess of electrons and protons in the expanding plume might be neutralizing highly charged analyte species.13 Recent studies have shown that both mechanisms might be taking place; however, the ratio of two ionization processes depends on experimental parameters, the nature of matrix and analyte.14

1.3 IR-MALDESI - one of the Softest Ionization Sources

In 2006, D. C. Muddiman and co-workers combined the properties of MALDI and ESI in one ambient ionization source – IR-MALDESI - which stands for infrared matrix-assisted laser desorption electrospray ionization (Figure 1.3).15-17 IR-MALDESI has several advantages over existing ionization sources. First, prior to analysis, sample undergoes no chemical modifications

(no extensive sample preparation steps)18; second, biologically-compatible ice is the only energy- absorbing matrix used during analyses17; third, ionization happens at ambient atmosphere; fourth, multiple- and singly-charged ions can be generated by the source19; and finally, IR-MALDESI tolerates samples with high salt concentrations20. In a typical IR-MALDESI experiment, the sample is sliced (10 µm < tissue thickness < 100 µm) and thaw-mounted onto a glass slide which is then placed onto translational stage. After a thin ice layer is formed on top of the sample, a mid-

IR laser pulse (2940 nm) excites O-H stretching modes of water which facilitate ablation of neutral species from the sample. These neutral molecules partition into charged ESI droplets and are ionized in an ESI-like manner. After ionization, a decreasing pressure gradient and voltage difference draw charged droplets/molecular ions into a mass spectrometer for analysis. The remote stage moves the sample in the x or y direction to ablate another set of tissue from an adjacent spot.18

1.4 IR-MALDESI Source Coupled with a Q Exactive Plus Mass Spectrometer

No matter what ionization method is used outside of a mass spectrometer, it is crucial to understand how the mass spectrometer works to be able to interpret collected data correctly. All of the data presented in this document was acquired using a Q Exactive Plus mass spectrometer, high-resolution accurate-mass (HRAM) mass spectrometer (Figure 1.4). After ionization in IR-

MALDESI source, ions pass through several transmission optics (S-lens and injection flatapole) where RF voltage gives rise to an electric field and focuses ions along the axis of the optics.

Figure 1.3. Schematic of IR-MALDESI.

Focused ions pass through the bent flatapole, a 90˚ arc, where RF voltage is applied to remove all residual solvent droplets and neutral molecules, while charged species enter the RF transfer multipole where ions with specified m/z values are selected and transferred into the C-trap. Ion populations in the C-trap are monitored by automatic gain control (AGC) target and injection time

(IT). Trapping too few or too many ions results in weak signal or detector oversaturation, respectively. AGC target maintains optimum number of ions for each scan, and IT is the time that ions are allowed to accumulate in the C-trap. AGC and IT are dependent parameters, and the one

Figure 1.4. Schematic of Q Exactive Plus coupled to IR-MALDESI. Image courtesy of Thermo Fisher Scientific. that is reached first controls ion populations in the C-trap. When ions reach the C-tap, they lose their kinetic energy upon collision with a nitrogen gas. After thermal cooling, ions can be transferred into an HCD (higher energy collisional dissociation) cell for fragmentation or directly injected into the Orbitrap mass analyzer for detection of intact molecules. In case of MS1, RF in the C-trap is ramped down and high DC voltage accelerates ions into the Orbitrap omitting HCD

10 cell. The Orbitrap consists of a center electrode surrounded by bell-shaped outer electrodes. When ions enter the Orbitrap, electric field is applied, and ions start rotating around the central electrode and oscillating along it. Image current from oscillated ions is measured by electron multiplier, digitized, and converted into frequency spectrum using fast Fourier transformation. Using a two- point calibration method, software converts frequency spectrum into a mass spectrum.

1.5 IR-MALDESI Mass Spectrometry Imaging

IR-MALDESI has been extensively used in mass spectrometry imaging (MSI) to monitor spatial distributions of different molecules in biological17,20-35 and non-biological samples36,37. In

MSI, one mass spectrum is collected from each voxel (volumetric element corresponding to one image pixel) in x and y directions. Untargeted mass spectrometry imaging (MSI) relies heavily on qualitative MS1 data because fragmentation of multiple features from the same voxel is impossible. Our current workflow in untargeted MSI analyses involves collection of MS1 data first, followed by atom counting and MS/MS on peaks that show interesting spatial distributions.

Using MSiReader38,39, an open source MSI software, one can generate heatmaps showing how ions of interest are distributed within a sample. Incorporation of the absolute quantification tool in

MSiReader39, now allows users to represent ion abundances in terms of absolute concentrations.

Also, when MS/MS data is unavailable, precursor peaks can be putatively annotated using the

METASPACE40 annotation engine, which reports lists with putative IDs for each m/z found in raw spectrum.

1.6 References

1. Budzikiewicz, H. & Grigsby, R. D. Mass spectrometry and isotopes: a century of research and discussion. Mass spectrometry reviews 25, 146-157 (2006).

2. Berglund, M. & Wieser, M. E. Isotopic compositions of the elements 2009 (IUPAC Technical Report). Pure and applied chemistry 83, 397-410 (2011).

3. Dole, M. et al. Molecular beams of macroions. The Journal of Chemical Physics 49, 2240- 2249 (1968).

4. Mack, L. L., Kralik, P., Rheude, A. & Dole, M. Molecular beams of macroions. II. The journal of chemical physics 52, 4977-4986 (1970).

5. Clegg, G. & Dole, M. Molecular beams of macroions. III. Zein and polyvinylpyrrolidone. Biopolymers: Original Research on Biomolecules 10, 821-826 (1971).

6. Iribarne, J. & Thomson, B. On the evaporation of small ions from charged droplets. The Journal of Chemical Physics 64, 2287-2294 (1976).

7. Yamashita, M. & Fenn, J. B. Electrospray ion source. Another variation on the free-jet theme. The Journal of Physical Chemistry 88, 4451-4459 (1984).

8. Yamashita, M. & Fenn, J. B. Negative ion production with the electrospray ion source. The Journal of Physical Chemistry 88, 4671-4675 (1984).

9. Fenn, J. B. Electrospray wings for molecular elephants (Nobel lecture). Angewandte chemie international edition 42, 3871-3894 (2003).

10. Cech, N. B. & Enke, C. G. Practical implications of some recent studies in electrospray ionization fundamentals. Mass spectrometry reviews 20, 362-387 (2001).

11. Tanaka, K. The origin of macromolecule ionization by laser irradiation (Nobel lecture). Angewandte chemie international edition 42, 3860-3870 (2003).

12. Knochenmuss, R. Ion formation mechanisms in UV-MALDI. Analyst 131, 966-986 (2006).

13. Karas, M., Glückmann, M. & Schäfer, J. Ionization in matrix‐assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors. Journal of mass spectrometry 35, 1-12 (2000).

14. Jaskolla, T. W. & Karas, M. Compelling evidence for lucky survivor and gas phase protonation: the unified MALDI analyte protonation mechanism. Journal of the American Society for Mass Spectrometry 22, 976-988 (2011).

15. Bokhart, M. & Muddiman, D. Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging analysis of biospecimens. Analyst 141, 5236-5245 (2016).

16. Nazari, M. & Muddiman, D. C. in Advances in MALDI and Laser-Induced Soft Ionization Mass Spectrometry 169-182 (Springer, 2016).

17. Robichaud, G., Barry, J. A. & Muddiman, D. C. IR-MALDESI mass spectrometry imaging of biological tissue sections using ice as a matrix. Journal of the American Society for Mass Spectrometry 25, 319-328 (2014).

18. Nazari, M., Bokhart, M. T. & Muddiman, D. C. Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI). Journal of visualized experiments: JoVE, e53942-e53942 (2016).

19. Sampson, J. S., Hawkridge, A. M. & Muddiman, D. C. Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry. Journal of the American Society for Mass Spectrometry 17, 1712-1716 (2006).

20. Ekelöf, M., McMurtrie, E. K., Nazari, M., Johanningsmeier, S. D. & Muddiman, D. C. Direct analysis of triterpenes from high-salt fermented cucumbers using infrared matrix- assisted laser desorption electrospray ionization (IR-MALDESI). Journal of the American Society for Mass Spectrometry 28, 370-375 (2017).

21. Robichaud, G., Barry, J. A., Garrard, K. P. & Muddiman, D. C. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging source coupled to a FT- ICR mass spectrometer. Journal of the American Society for Mass Spectrometry 24, 92- 100 (2013).

22. Barry, J. A., Groseclose, M. R., Robichaud, G., Castellino, S. & Muddiman, D. C. Assessing drug and metabolite detection in liver tissue by UV-MALDI and IR-MALDESI mass spectrometry imaging coupled to FT-ICR MS. International journal of mass spectrometry 377, 448-455 (2015).

23. Barry, J. A. et al. Mapping antiretroviral drugs in tissue by IR-MALDESI MSI coupled to the Q Exactive and comparison with LC-MS/MS SRM assay. Journal of the American Society for Mass Spectrometry 25, 2038-2047 (2014).

24. Nazari, M. & Muddiman, D. C. Polarity switching mass spectrometry imaging of healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Analyst 141, 595-605 (2016).

25. Nazari, M. & Muddiman, D. C. Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling. Analytical and bioanalytical chemistry 407, 2265-2271 (2015).

26. Rosen, E. P., Bokhart, M. T., Ghashghaei, H. T. & Muddiman, D. C. Influence of desorption conditions on analyte sensitivity and internal energy in discrete tissue or whole body imaging by IR-MALDESI. Journal of the American Society for Mass Spectrometry 26, 899-910 (2015).

27. Sampson, J. S., Murray, K. K. & Muddiman, D. C. Intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to FT-ICR mass spectrometry. Journal of the American Society for Mass Spectrometry 20, 667-673 (2009).

28. Meier, F., Garrard, K. P. & Muddiman, D. C. Silver dopants for targeted and untargeted direct analysis of unsaturated lipids via infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI). Rapid Communications in Mass Spectrometry 28, 2461-2470 (2014).

29. Bokhart, M. T. et al. Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization. Analytical and bioanalytical chemistry 407, 2073-2084 (2015).

30. Thompson, C. G. et al. Mass spectrometry imaging reveals heterogeneous efavirenz distribution within putative HIV reservoirs. Antimicrobial agents and chemotherapy, AAC. 04952-04914 (2015).

31. Nazari, M. et al. Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI. Analytical and bioanalytical chemistry 410, 953-962 (2018).

32. Nazari, M., Bokhart, M. T., Loziuk, P. L. & Muddiman, D. C. Quantitative mass spectrometry imaging of glutathione in healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Analyst 143, 654-661 (2018).

33. Rosen, E. et al. in Conference on Retroviruses and Opportunistic Infections 2015.

34. Thompson, C. et al. Mass Spectrometry Imaging of Hair Strands Allows for Evaluation of Long Term Antiretroviral Adherence. AIDS research and human retroviruses 30, A69- A69 (2014).

35. Rosen, E. P. et al. Analysis of antiretrovirals in single hair strands for evaluation of drug adherence with infrared-matrix-assisted laser desorption electrospray ionization mass spectrometry imaging. Analytical chemistry 88, 1336-1344 (2015).

36. Cochran, K. H., Barry, J. A., Robichaud, G. & Muddiman, D. C. Analysis of trace fibers by IR-MALDESI imaging coupled with high resolving power MS. Analytical and bioanalytical chemistry 407, 813-820 (2015).

37. Cochran, K. H., Barry, J. A., Muddiman, D. C. & Hinks, D. Direct analysis of textile fabrics and dyes using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry. Analytical chemistry 85, 831-836 (2012).

38. Robichaud, G., Garrard, K. P., Barry, J. A. & Muddiman, D. C. MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform. Journal of the American Society for Mass Spectrometry 24, 718-721 (2013).

39. Bokhart, M. T., Nazari, M., Garrard, K. P. & Muddiman, D. C. MSiReader v1. 0: evolving open-source mass spectrometry imaging software for targeted and untargeted analyses. Journal of the American Society for Mass Spectrometry 29, 8-16 (2018).

40. Palmer, A. et al. FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry. Nature methods 14, 57 (2017).

CHAPTER 2

Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer

using Isotope Ratio Mass Spectrometry

Reprinted with permission from: Khodjaniyazova, S.#, Nazari, M.#, Garrard, K. P., Matos, M. P., Jackson, G. P., and Muddiman, D. C. Analytical Chemistry, 2018, 90(3), 1897-1906. #Authors contributed equally to this work. Copyright © 2018, American Chemical Society.

2.1 Introduction

Accurate determination of elemental compositions is one of the most challenging aspects in untargeted metabolomics analyses.1 High resolving power coupled with high mass measurement accuracy (MMA) alone cannot be used for confident identification of unknowns2-5 because, even at high MMA (<1 ppm), several elemental compositions are possible.5-7 However, high MMA combined with spectral accuracy (SA) can often lead to elucidation of a single elemental composition and/or confirmation of a database hit.6, 8In fact, using solely isotopic distributions can remove >95% of false candidates.6 Spectral accuracy is the ability of the mass analyzer to accurately measure isotopic distributions (including isotopic fine structures) which, when coupled with high MMA, can be used for estimating the number of specific elements in an unknown.4, 5, 7-

15 For example, to estimate the number of carbon atoms in a molecule, the relative abundance of

13 13 A+1 ( C1) peak is divided by the natural abundance of C on Earth (∼1.11%) based on natural abundance values reported by the National Institute of Standards and Technology (NIST) or

International Union of Pure and Applied Chemistry (IUPAC).16 However, the values in these libraries are presented as “best-measurement” values, whereas the true isotopic abundances fall somewhere within the observed range of natural variations.16 For instance, the relative abundance

16 of 13C in terrestrial matter actually ranges from 0.96% to 1.15%, which provides sufficient variance to cause incorrect assessments of the number of carbons in a molecule.16

The motivation of the present work is to characterize SA of the Orbitrap mass analyzer2, 17-

19 based on the absolute ion abundances to allow its use in untargeted mass spectrometry imaging

(MSI) studies with infrared matrix-assisted laser desorption electrospray ionization (IR-

MALDESI)20, 21 coupled to the Thermo Fisher Scientific Q Exactive Plus mass spectrometer.22 IR-

MALDESI is an ambient ionization source where a mid-IR laser is used to desorb neutral species from a sample. The desorbed materials partition into the charged droplets of an orthogonal electrospray, where ions are formed in an ESI-like fashion.20 These ions are next stored in the C- trap for a predetermined amount of time, denoted by the maximum injection time (IT),23 and the automatic gain control (AGC) function in the Q Exactive Plus mass spectrometer is disabled due to the pulsed nature of the IR-MALDESI source. While MS/MS analyses can be performed on peaks of interest to confirm the structure of putative identifications,22 acquiring MS/MS spectra for every analyte in every voxel is virtually impossible in untargeted MSI analyses. Therefore, identification of unknowns in untargeted IR-MALDESI studies relies solely on MMA and SA.

Nazari et al. have previously demonstrated the utility of SA and sulfur counting in the identification of metabolites in an untargeted polarity switching MSI analysis,7 where analyte was confidently identified from four potential candidates generated in MELTIN database.24

One critically important and limiting factor in accurately characterizing the SA of a mass analyzer is the fact that there is a variation in the relative abundance of different isotopes of each element found in nature. Thus, to investigate the effects of absolute ion abundance on the

Orbitrap’s ability to recover expected isotopic distributions, we characterized known compounds spanning a wide mass range using isotope ratio mass spectrometry spectrometry (IRMS) to

17 accurately measure abundance of stable isotopes. Results from IRMS analyses allowed us to

13 15 34 precisely monitor changes in relative abundances of C1, N1, and S1 molecular ions measured using the Q Exactive Plus. We report two experimentally determined optimal conditions for each of three arbitrarily defined mass windows of small (100 < MW (Da) < 400), medium (400 < MW

13 15 (Da) < 900), and large (1000< MW (Da) < 1500) compounds: (1) optimum absolute C1, N1,

34 and S1 ion abundance for accurate carbon, nitrogen, and sulfur counting, respectively, and (2) thresholds for the absolute monoisotopic ion abundances required for high SA. In the work presented here, we demonstrate that it is crucial to establish thresholds for absolute ion abundances because changes in the absolute ion abundance could influence the MMA and SA and subsequently hinder the ability to confidently identify unknown analytes. Establishing the optimum absolute ion abundances required for high MMA and SA will help to reduce the number of potential identifications (IDs), generated by searching the accurate mass in databases such as METLIN24 or

HMDB,25 because compounds whose absolute ion abundances are above certain thresholds can be used for confident estimation of elemental compositions. Once a narrow list of potential IDs is generated, a different algorithm can be used to predict the elemental compositions of those compounds that pass the first screening.4, 7

2.2 Experimental Section

2.2.1 Materials

Caffeine was purchased from both Sigma-Aldrich (St. Louis, MO) and the International

Atomic Energy Agency (IAEA). The two caffeine samples are termed caffeineSigma and caffeineIAEA, respectively. The tetrapeptide MRFA acetate salt and acetic acid were also purchased from Sigma-Aldrich. HPLC-grade methanol, water, and acetonitrile were purchased from Burdick

& Jackson (Muskegon, MI). Ultramark was purchased from ABCR GmbH (Karlsruhe, Germany).

The isotope standards of glutamic acid (USGS-40 and USGS-41) were obtained from the

United States Geological Survey (USGS, Reston, VA). An isotope standard of sulfanilamide was purchased from IVA Analysentechnik e. K. (Meerbusch, Germany).

2.2.2 Direct Infusion of Caffeine, MRFA, and Ultramark Mixture Using Q Exactive Plus

In Q Exactive Plus analyses, a mixture of caffeineSigma, MRFA, and ultramark was ionized in the ESI interface. Neutral species were filtered in the bent flatapole whereas ions were efficiently transferred to the RF-only quadrupole mass filter. Exiting the quadrupole, ions were cooled in the

C-trap26 and injected into the Orbitrap for further image current detection and fast Fourier (FT) transformation.27

The Q Exactive Plus mass spectrometer was calibrated prior to analyses. CaffeineSigma,

MRFA, and ultramark were dissolved in 5 mL of acetonitrile, 4.68 mL of 50:50 methanol:water

(v/v), and 100 μL of acetic acid to yield a mixture containing 2 μg/mL caffeine, 0.7 μg/mL MRFA, and 18 μg/mL ultramark. The mixture was directly infused into the Q Exactive Plus mass spectrometer using ESI at a flow rate of 2 μL/min with the electrospray voltage and inlet temperature at 4.0 kV and 320 °C, respectively. The mixture was analyzed from m/z 150 to m/z 2000 with a resolving power of 140 000fwhm at m/z 200. Ninety-nine transient scans (1 microscan each) were recorded at each of eight AGC targets: 2 × 104, 5 × 104, 1 × 105, 2 × 105, 5

× 105, 1 × 106, 3 × 106, and 5 × 106. The maximum IT for each injection was set to 100 ms to provide enough time for ion accumulation at high AGC targets. To achieve low ppm MMA, peaks of diisooctyl phthalate at m/z 391.2843 [M + H+]+ and 413.2662 [M + Na+]+ were used as lock- masses for internal calibration.28

2.2.3 Direct Infusion of IRMS Reference Standards Using Q Exactive Plus

Each isotope reference standard was diluted in 5 mL of acetonitrile (ACN), 5 mL of 50:50

19 methanol:water (v/v), and 100 μL of acetic acid to have four standards for direct infusion analysis:

0.03 mg/mL glutamic acid 1 (USGS-40), 0.002 mg/mL caffeineIAEA (IAEA-600), 0.002 mg/mL sulfanilamide, and 0.03 mg/mL glutamic acid 2 (USGS-41). Glutamic acid 1, caffeineIAEA, sulfanilamide, and glutamic acid 2 were directly infused one at a time into the Q Exactive Plus mass spectrometer in the listed order. A relatively narrow m/z range of 70–280 was measured to make sure that the mass of each reference standard fell roughly in the middle of selected m/z range.4 The AGC target and maximum IT were set to 1 × 106 and 300 ms, respectively, and 100 transient scans (1 microscan each) were obtained per analyte at a resolving power of 140 000fwhm at m/z 200.

Even though analysis of narrow m/z ranges29 with increased number of microscans30 at higher RPs can improve SA, we used instrumental parameters that are more typical in untargeted

MSI experiments (e. g., wide m/z range, 1 microscan, RP = 140 000 fwhm at m/z 200) to demonstrate the applicability of this approach to more realistic untargeted IR-MALDESI MSI studies.

2.2.4 Analysis of CaffeineSigma, MRFA, and Ultramark Using IRMS

For the bulk isotope analysis of δ13C and δ15N, samples of approximately 0.5 mg of caffeineSigma, 0.3 mg of MRFA, and 0.9 mg of ultramark were weighed in tin capsules and placed in a Thermo Flash HT Plus elemental analyzer (EA) coupled via a Conflo IV interface (Thermo

Finnigan, Waltham, MA) to a Thermo Delta V Advantage isotope ratio mass spectrometer. The elemental analyzer converted each sample (caffeineSigma, MRFA, or ultramark) into simple fixed gases (e.g., N2, CO2) using the standard combustion and a reduction reactor, followed by separation in a packed gas chromatography (GC) column using helium (Matheson, Fairmont, WV) as the carrier gas. The purified gas molecules were subsequently ionized via electron ionization (EI), and

20 the abundances of these ionized gases were detected simultaneously using multiple Faraday cups after passing through the magnetic sector mass analyzer.31 Data acquisition was carried out using

Isodat 3.0 software (Thermo Finnigan, Waltham, MA).

Carbon isotope ratios were measured relative to a compressed reference CO2 gas (Airgas,

Morgantown, WV) and normalized to the international scale relative to Vienna Pee Dee Belemnite

(VPDB) using a two-point linear regression calibration based upon the certified reference materials

USGS-40 (−26.39 ‰) and USGS-41 (+37.63 ‰).32 The correction for 17O was performed using the standard Santrock algorithm.33 For nitrogen isotope ratios, delta values were measured relative to compressed nitrogen (Airgas, Morgantown, WV) and were normalized to international air

N2 using a two-point calibration curve composed of USGS-40 (−4.52 ‰) and USGS 41 (+47.57

‰). Triplicate measurements of each sample provided mean values and 95% confidence intervals, which were reported on the per mill (‰) scale relative to VPDB for δ13C and air for δ15N.

Sulfur bulk isotope ratios of MRFA samples were measured by the United States Geological

Survey (Reston Stable Isotope Laboratory, Reston, VA) using a continuous flow isotope ratio mass spectrometer.34 Results were reported in per mill (‰) relative to Vienna-Canyon Diablo Troilite

(VCDT) and defined by assigning an exact value of −0.3‰ to IAEA-S-1 (silver sulfide),35 and no correction was conducted for oxygen isotopic composition.

2.2.5 Data Analysis

To correctly assign peaks in isotopic distributions measured using the Q Exactive Plus, theoretical exact molecular masses were calculated using exact atomic mass values reported by

IUPAC.16 To avoid confusion, we refer to ultramark as ultramark 1421 throughout this manuscript because in data analysis we focused only on the most abundant [M + H+]+ ion at m/z 1421 (Figure

A.1). The .RAW files generated by the Q Exactive Plus were processed in XCalibur software

(version 2.2, Thermo Fisher Scientific, San Jose, CA) and then converted into the .mzML format using the open-source MSConvertGUI tool from ProteoWizard.29 Subsequently, the m/z values within ±2.5 ppm tolerance were extracted using the RawMeat tool (version 2.1, VAST Scientific,

Cambridge, MA) and exported into Excel. Masses for isotopologues were calculated using the difference in their exact masses from the monoisotopic peak. Extracted ion chromatograms (XIC) for each peak (±2.5 ppm) were generated using XCalibur and exported into Excel. We exported

IT for each transient scan to calculate the absolute ion abundances (abundance from .RAW file [in ions/seconds] × IT [in seconds]) because prescanned AGC targets result in slightly different ITs from scan to scan. All subsequent analyses and calculations were performed in Excel.

2.3 Results and Discussion

2.3.1 Orbitrap’s Sensitivity of Measuring Relative Abundances across Eight AGC Targets

We characterized the Orbitrap’s performance in a Q Exactive Plus mass spectrometer at a resolving power of 140 000fwhm at m/z 200 by altering AGC targets to monitor how different ion populations affect mass and spectral accuracy in arbitrarily defined small (100 < MW (Da) < 400), medium (400 < MW (Da) < 900), and large (1000< MW (Da) < 1500) compounds represented by caffeineSigma (C8H10N4O2), MRFA (C23H37N7O5S), and ultramark (C28H18O6N3P3F24(C2F4)n, n =

0–12). The effects of varying the resolving power were not investigated in this study because this topic has already been discussed in detail by others.4, 9, 10 We prepared a homemade positive ion calibration solution from caffeine, MRFA, and ultramark to have materials from the same lot numbers for IRMS analyses. The homemade calibration solution had smaller concentrations of caffeineSigma (2 μg/mL) and MRFA (0.7 μg/mL) relative to commercial Thermo Scientific Pierce

LTQ ESI Positive Ion Calibration Solution, which usually has caffeine and MRFA concentrations of 20 μg/mL and 1 μg/mL, respectively. The full range spectrum in Figure A.1 shows that the

22 relative abundances in homemade calibration solution differ quite substantially from the relative abundances normally seen in the spectrum of the commercially available calibration solution.

As mentioned above, SA in tandem with high MMA can be used for confident elucidation of unknown analytes. As expected, in lock-mass controlled analyses, MMAs for caffeineSigma,

MRFA, and ultramark fell within the accepted range of ±2.5 ppm across all eight AGC targets.

Moreover, with larger ion populations, MMA for caffeineSigma, MRFA, and ultramark approached

∼0.6 ppm, ∼0.7 ppm, and ∼0.6 ppm, respectively (Figure A.2). However, MMA alone cannot be used for accurate identification of unknown compounds with complex elemental compositions.6

Once it was shown that the MMA was preserved at all ion populations, we turned our attention to characterizing how accurately the Orbitrap measures relative abundances of stable isotopologues

13 15 34 such as C1, N1, and S1 that can be used in carbon, nitrogen, and sulfur counting, respectively.

To ensure the most accurate characterization of the Orbitrap’s SA, the relative abundances of the stable isotopes in caffeineSigma, MRFA, and ultramark were measured using EA-IRMS. The values were reported using the δ notation in units of parts per thousand, or per mill (‰). δ stands for the difference in isotopic composition of the analyte relative to that of the reference standard.31

The same compounds (caffeineSigma, MRFA, and ultramark) from the same lot numbers were mixed together and directly infused into the Q Exactive Plus mass spectrometer by ESI and analyzed at the eight different AGC targets. Knowing the elemental composition of each of the three compounds, we were able to convert abundances measured with Q Exactive Plus into atom percent (isotope’s natural abundance) and calculate the δ values to monitor how spectral accuracy changed across eight AGC targets. First, the abundances (from .RAW files) measured using the

Orbitrap were used to calculate the abundance ratios of the heavier isotope (e.g., 13C) to the lighter

23 isotope (e.g., 12C) of each atom in a given analyte. The ratios were then normalized to the number of atoms as shown in Equation 2.1:

퐚퐛퐮퐧퐝퐚퐧퐜퐞 퐨퐟 퐀+ퟏ 퐑 = (Equation 2.1) 퐬퐚퐦퐩퐥퐞 (퐚퐛퐮퐧퐝퐚퐧퐜퐞 퐨퐟 퐀) × 퐧퐨. 퐨퐟 퐚퐭퐨퐦퐬

Rsample ratios were then used to calculate the atom percent (atom %) and δ values for each atom according to Equations 2.2 and 2.3, respectively:

R atom% = sample × 100 (%) (Equation 2.2) 1+ Rsample

R 훿 = ( sample − 1) × 1000 (‰) (Equation 2.3) Rstandard

Rstandard in Equation 2.3 represents the same ratio as Rsample but for a standard reference material that is naturally enriched in stable isotopes. Ion abundances measured in the Orbitrap were

13 converted into δOrbitrap and plotted against absolute ion abundances of C1 isotopologues for all three compounds as shown in Figure 2.1. The mean δexpected values and their corresponding 95% confidence intervals, measured using IRMS, are shown on the top right of the Figure 2.1. Mean

δexpected values are also depicted as solid lines on the graph for easier visualization. It can be seen

13 13 that the calculated δOrbitrap C for caffeineSigma, MRFA, and ultramark 1421 approach δexpected C as

13 the absolute ion abundances increase; however, even with large ion populations, δOrbitrap C does

13 15 34 not fall within the 95% confidence interval of δexpected C. The values for δOrbitrap N and δOrbitrap S distributions in caffeineSigma and MRFA follow the same trend (Figure A.3).

To further investigate the ability of the Orbitrap to accurately measure isotope ratios at higher ion populations, four IRMS reference standards were directly infused into the Q Exactive

Plus mass spectrometer individually and analyzed from m/z 70 to m/z 280 to have all four peaks fall roughly in the middle of the selected mass range (Figure 2.2). The key step in this analysis was injection of each reference standard one at a time with the prescanned AGC target and fixed

13 13 Figure 2.1. Dependence of calculated δOrbitrap C on absolute ion abundance of A+1 ( C1) peak 13 using caffeineSigma, MRFA, and ultramark 1421. δexpected C values with 95% confidence interval, as measured by IRMS, are shown on the top right and are also depicted as solid lines on the figure. The inset on top shows the zoomed-in region of -100 to 100 on the y-axis. Each data point represents a single transient scan. maximum IT set to 1 × 106 ions and 300 ms, respectively. One hundred consecutive spectra were recorded to establish the mean and 95% CI for the reported isotope ratios. As shown in Table 2.1,

13 15 δexpected C and δexpected N in caffeineIAEA, glutamic acid 1, and glutamic acid 2 are the only

13 expected values to fall within the confidence interval of the measured isotope ratios (δOrbitrap C

15 and δOrbitrap N). The rest of the δexpected values do not fall within the confidence interval of the

13 calculated δOrbitrap values. As we predicted, all the δOrbitrap C values measured with the Orbitrap

13 have much lower precision in comparison to δexpected C measured using IRMS. These results suggest that even at seemingly optimized parameters that result in ideal MMA, the Q Exactive

Plus mass spectrometer cannot measure 13C/12C, 15N/14N, and 34S/32S isotope ratios as accurately and precisely as isotope ratio MS (IRMS).

Figure 2.2. Orbitrap mass spectra collected in positive ESI mode at RPfwhm of 140 000 at m/z 200 for (A) glutamic acid 1 (USGS-40), (B) caffeineIAEA (IAEA-600), (C) sulfanilamide, and (D) glutamic acid 2 (USGS-41) that were injected separately one sample at a time. User-defined AGC target for each injection was set to 1 × 106 and acquired m/z range was 70–280. Instrument- determined average injection time (n = 100 transient scans) for glutamic acid 1, caffeineIAEA, sulfanilamide, and glutamic acid 2 was 4, 21, 14, and 4 ms, respectively.

The typical precision (95% CI) of IRMS measurements is on the order of 0.2‰ for carbon and 0.3‰ for nitrogen, whereas the Orbitrap provided pooled confidence intervals on the order of

9‰ for carbon and 17‰ for nitrogen. The Orbitrap is therefore approximately 50 times less precise than the IRMS. Heisenberg’s uncertainty principle implies that the amplitude and the frequency of an image current in the Orbitrap cannot simultaneously be known to high degrees of confidence, and that the better one knows the time domain (i.e., the frequency, and therefore the m/z), the worse one knows the amplitude, or ion abundance.36 Miladinovic et al. have shown that FT-ICRs are capable of precisions on the order of 1 ppm in the time domain but at the expense of only ∼1% precision in the abundance domain.37 The main advantage of multicollector magnetic sector

Table 2.1. Analysis of Four IRMS Reference Standards Using a Q Exactive Plus.a

IRMS Standards isotopes δOrbitrap δexpected

15N -38 ± 48 (SD) +1.0 ± 0.2 (SD) caffeine (IAEA-600) 13C -30 ± 19 (SD) -27.77 ± 0.04 (SD)

15N -80 ± 76 (SD) -4.52 ± 0.06 (SD) glutamic acid 1 (USGS-40) 13C -7 ± 21 (SD) -26.39 ± 0.04 (SD)

15N -19 ± 81 (SD) +47.6 ± 0.1(SD) glutamic acid 2 (USGS-41) 13C +51 ± 20 (SD) +37.63 ± 0.05 (SD)

15N -130 ± 24 (95%CI) -0.4 ± 0.2 (95%CI)

sulfanilamide 13C -79 ± 10 (95%CI) -27.8 ± 0.3 (95%CI)

34S -51 ± 14 (95%CI) +19.0 ± 0.7 (95%CI)

a 15 13 34 Abundances of ions of interest ( N1, C1, and S1) collected using the Q Exactive Plus were converted to δOrbitrap and compared to the expected values (δexpected) measured using IRMS. instrument comes from the ability to measure isotope abundances simultaneously on at least two detectors and for extended durations. These two features compensate for ion source variation over time and enable more signal averaging, which both lead to better precision. After obtaining such high precision, one must only then correct for any bias in the instrument and detection system, which is readily accomplished through comparison of unknowns to isotope standards. Also, in

IRMS analyses, each sample is converted into simple pure gases before analysis and thus abundances of isotopes are measured on the atomic level. In contrast, the Orbitrap measures relative abundances of molecular ions with different isotopic compositions.18, 38, 39 Therefore, in

FT-based measurements, such as the Orbitrap, it is oftentimes not possible to independently measure abundances of all the contributing isotopic ions, so approximations and assumptions must

27 be made regarding the abundance of unresolved isotopes. For instance, the approximated expected

13 relative abundance of A+1 ( C1) will always be slightly larger than the true relative abundance of

13 A+1 ( C1) peak because expected relative abundance of A+1 is the product of the number of atoms and the atom percent of 13C from all isotopologues.

2.3.2 Orbitrap’s Sensitivity of Carbon Counting across Eight AGC Targets

15 13 34 To calculate the expected relative abundance of N1, C1, and S1 peaks, accurate atom percent values obtained from IRMS were multiplied by the total number of atoms of interest in a molecule. Because IRMS measures the abundance ratio of one heavy (e.g., 13C) to one light isotope

(e.g., 12C), we used Equation 2.4 for computing the binomial probability distribution to estimate

13 the abundance of the A+2 peak in a molecule with a total of N atoms of interest (e.g., C2 peak):

푁! 푃(푛) = 푝푛(1 − 푝)푁−푛 (Equation 2.4) 푛!(푁−푛)!

In Equation 2.4, n is the isotopic peak (n = 0 being monoisotopic peak A and n = 1 is the

A+1 peak, etc.), N is the total number of atoms of interest, and 0 < p < 1 is the relative abundance

(RA) of the stable isotope. The expected relative abundance of each n > 1 peak was calculated using Equation 2.5:

푃(푛) 푒푥푝푒푐푡푒푑 푅퐴 = × 100% (Equation 2.5) 푃(0)

Figure 2.3 shows representative mass spectra for ultramark 1421, caffeineSigma, and MRFA collected with Q Exactive Plus at a resolving power of 140 000fwhm at m/z 200 and AGC targets of

2 × 105, 5 × 105, and 3 × 106, respectively. At these AGC targets all three compounds have relative abundances that best match their expected relative abundances depicted as red dots and calculated

18 using δ values from IRMS analyses. Note that in Figure 2.3A there is no red dot above O1 peak because we measured only carbon and nitrogen isotope ratios in caffeineSigma. It can be seen that

13 15 C1 and N1 peaks are only baseline resolved in caffeineSigma, whereas in heavier molecules such

15 13 as ultramark 1421, the N1 peak is not resolved from the C1 peak. In addition to the A+1 peaks,

18 13 34 13 the A+2 peaks of caffeineSigma ( O1 and C2) and MRFA ( S1 and C2) are also resolved from each other. Figure 2.3 suggests that in general the Orbitrap slightly underestimates relative abundances of heavier isotopic species in isotopic distributions, specifically ∼12%

Figure 2.3. Representative Orbitrap mass spectra for (A) caffeineSigma, (B) MRFA, and (C) ultramark 1421 collected in positive ESI mode at RPfwhm of 140 000 at m/z 200 and AGC targets of 5 × 105, 3 × 106, and 2 × 105, respectively. Each spectrum was acquired within m/z range of 150–2000. Instrument-determined average injection times (n = 99 transient scans) for caffeineIAEA, MRFA, and ultramark 1421 was 1, 6, and 0.3 ms, respectively.

13 34 underestimation for C2 in caffeine and ∼15% for S1 in MRFA. According to Su et al., this underestimation could be a result of interference between isotopic species of similar masses that oscillate at almost identical frequencies in the Orbitrap.9

To estimate the number of carbons in an unknown molecule, the relative abundance of A+1

13 13 ( C1) peak is divided by the natural abundance of C. Most often the “best measurement” value from a database such as IUPAC is used for this process.8 However, using abundances from such databases for carbon counting does not result in accurate estimation of elemental compositions because not all compounds have the same relative 13C abundance. For instance, the best measurement for 13C atom percent from IUPAC is 1.108%, while in our analysis the atom percent values measured using IRMS are 1.070%, 1.086%, and 1.061% for caffeineSigma, MRFA, and ultramark 1421, respectively (Figure A.4). We used EA-IRMS atom percent values to measure how well the Q Exactive Plus is capable of counting carbons, nitrogens, and sulfurs when the atom percent is “known”. Using Equation 2.6, we calculated the difference between known and observed number of carbons across different ion populations as shown in Figure 2.4:

13 13 (푘푛표푤푛 푛표. 표푓 푐푎푟푏표푛푠) × ( 퐶1 푎푡표푚% ) − (표푏푠푒푟푣푒푑 푅퐴 표푓 퐶1) 푘푛표푤푛 − 표푏푠푒푟푣푒푑 푛표. 표푓 푐푎푟푏표푛푠 = 13 퐶1 푎푡표푚% (Equation 2.6)

Atom percent values in Equation 2.6 were determined using EA-IRMS and observed relative abundances (RA) were measured on the Q Exactive Plus. Using Equation 2.6 with atom percent values from IRMS, we monitored how SA and thus Orbitrap’s ability to count carbons

(Figure 2.4), nitrogens (Figure A.5A), and sulfurs (Figure A.5B) was affected across eight AGC targets. Note that at smaller AGC targets, nitrogen in caffeineSigma (Figure A.5A) and sulfur in

MRFA (Figure A.5B) were not resolved, and that is why there are seven and six data points in caffeineSigma and MRFA plots, respectively. Figures 2.4, A.5A, and A.5B demonstrate that the

30 difference between known and expected number of atoms follows the same trend in each of the three compounds: the smallest difference is achieved roughly in the middle of the absolute ion abundance curve, and the larger difference is observed at the lowest and highest absolute ion abundances. It is worth noting that while the mean difference gets worse at high absolute ion abundances for MRFA (Figure 2.4), the precision of the measurement is still improved significantly. Moreover, Figure A.6 shows how the difference between expected and observed

13 relative abundance of C2 peak in MRFA improves at higher AGC targets. Note that at the

4 13 smallest AGC target of 2 × 10 , the C2 peak was indistinguishable from the signal-to-noise, and

4 13 at AGC target of 5 × 10 , most of the C2 peaks had signal intensity of zero. IRMS does not

13 measure relative abundance of C2 isotopologues, so in the probability equation we were

13 13 estimating abundance of C2 in MRFA based on an already known atom percent of C from

13 IRMS analysis. Relative abundance of C2 peak cannot be used in carbon counting, and this explains why we were mainly focusing on A+1 species.

“The highest ion population” in this study refers to the absolute ion abundance at the maximum AGC target (5 × 106), but more ions can be stored in the Orbitrap by disabling AGC target and setting maximum IT to a high number (e.g., 100 ms). However, when too many ions are injected into the Orbitrap (e.g., IT ≥ 100 ms), molecular ions start experiencing space-charge effects, which result in suppressed abundance signals in lighter molecules and enhanced abundance signals in heavier molecules. Spectral accuracy is the most affected in the least abundant species because species with smaller ion cloud densities have much faster decay rates.40 When comparing

MS spectra of caffeineSigma, MRFA, and ultramark 1421 collected at prescanned AGC targets of 5

× 105, 3 × 106, and 2 × 105, respectively, with spectra collected at IT of 100 ms with fixed AGC function, the absolute ion abundances of monoisotopic peaks shown in Table 2.2 changed

5 6 5 significantly. At AGC targets of 5 × 10 , 3 × 10 , and 2 × 10 , caffeineSigma, MRFA, and ultramark

1421, respectively, have relative abundances that best match the expected relative abundances measured using IRMS. Also, each of these AGC targets has maximum IT that is much shorter than

100 ms Table 2.2 shows that at longer ITs, the absolute monoisotopic ion abundance decreased by 2 orders of magnitude in caffeineSigma, did not change much in MRFA, and increased by 2 orders

13 of magnitude in ultramark 1421. As a result, relative abundance of A+1 ( C1) peak in caffeine was reduced by a factor of ∼2 from 8.58% to 4.85%, while the expected relative abundance was

8.56% according to IRMS measurements. This was not the case for heavier molecules such as

MRFA and ultramark 1421, which did not have significant changes in spectral accuracy at IT of

Figure 2.4. Deviations from the known number of carbons across different ion populations in caffeineSigma, MRFA, and ultramark 1421. Atom percent values, used in carbon counting, were obtained from IRMS analyses. Error bars correspond to 95% confidence interval of the mean (n = 99).

Table 2.2. Comparison of Absolute Monoisotopic Ion Abundances Acquired at Small (column 2) and Large (column 3) Total Ion Populationsa)

absolute ion abundance of A peak at absolute ion abundance of A peak optimum AGC targets at IT = 100 ms 5 5 3 caffeineSigma 2.1 × 10 ions (AGC = 5 × 10 ) 2.3 × 10 ions MRFA 1.8 × 104 ions (AGC = 3 × 106) 3.0 × 104 ions ultramark 1421 5.6 × 103 ions (AGC = 2 × 105) 2.6 × 105 ions a) Note that at “optimum AGC targets”, shown in column 2, experimental relative abundances (Orbitrap) match the expected relative abundances (IRMS) the best.

34 100 ms: the relative abundance of A+2 ( S1) peak in MRFA had an insignificant change from

3.76% (IT = 6.09 ms) to 3.75% (IT = 100 ms) while the expected relative abundance was 4.33%.

13 Similarly, in ultramark 1421, observed relative abundance of C1 peak changed slightly from

30.28% (IT = 0.33 ms) to 29.81% (IT = 100 ms) while expected relative abundance was 29.70%.

Figures 2.4, A.5A, and A.5B confirm that indeed there is an optimum number of injected ions that results in sufficient spectral accuracy to allow for accurate estimation of the number of carbons, nitrogens, and sulfurs. To accurately estimate the number of carbons with the desired

13 3 precision of less than ±0.5 atoms, the absolute C1 ion abundance should be ∼7.9 (±0.15) × 10 in

3 4 caffeineSigma (±0.12 atoms), ∼ 1.1 (±0.04) × 10 in MRFA (±0.11 atoms), and ∼1.5 (±0.03) × 10 in ultramark 1421 (±0.45 atoms) (Figure 2.4). It is worth noting that optimum absolute ion abundance of MRFA peptide with charge state of +1 is 1 order of magnitude smaller than in ultramark 1421 and almost identical to abundance threshold in caffeineSigma. This is explained by

MRFA’s ionization efficiency at +1 charge state. Molecular ions of MRFA with charge state of +2 are much more abundant than ultramark 1421 and less abundant than caffeineSigma. Reliable

15 nitrogen counting (±0.31 nitrogens) in caffeineSigma is possible at absolute N1 ion abundance of

∼3.4 (±0.22) × 102 (Figure A.5A), and reliable sulfur counting (±0.13 sulfurs) in MRFA can be

34 2 achieved when absolute S1 ion abundance is ∼6.6 (±0.31) × 10 (Figure A.5B). Interestingly,

33 when counting atoms with atom percent values from IUPAC at above-mentioned thresholds, we can still accurately estimate number of carbons (±0.39 atoms, Figure A.7) and nitrogens (±0.35 atoms, Figure A.5A) in caffeineSigma, and sulfurs in MRFA (±0.10 atoms, Figure A.5B).

However, as expected, when counting carbons in MRFA and ultramark 1421 using atom percent values from IUPAC, the tolerance lowers to ±0.56 and ±0.76 atoms in MRFA and ultramark 1421, respectively. It is worth noting that, because the relative abundance of 34S in nature is much higher than that of 13C and 15N, the tolerance window for sulfur counting can be wider compared to the tolerances used for carbon and nitrogen counting.

To estimate the minimum absolute monoisotopic ion abundance required for high spectral accuracy, we generated the Pearson chi-square (χ2) distributions across eight AGC targets for each of the three test compounds (Figure 2.5). To calculate χ2 values for relative abundances we used

Equation 2.7:

(푂푏푠푒푟푣푒푑 푅퐴 − 퐸푥푝푒푐푡푒푑 푅퐴)2 χ2 = ∑ (Equation 2.7) 퐸푥푝푒푐푡푒푑 푅퐴

χ2 for each compound was calculated by summing the χ2 of an individual atom in the compound, depending on peak resolution: 2 2 2 2 χCaffeine(Sigma) = χC + χN + χO 2 2 2 χMRFA = χC + χS χ2 = χ2 Ultramark 1421 C The calculated χ2 values fall within a range of 0 < χ2 < ∞, with smaller values indicating higher spectral accuracy. In Figure 2.5, χ2 values are distributed in distinct packets because at each

AGC target only a restricted number of ions are stored in the C-trap. It can be seen that the SA of caffeineSigma, MRFA, and ultramark 1421 improves as the absolute ion abundance of the monoisotopic peak increases, presumably because more ions are stored in the C-trap. It should be noted that there is an upper limit to the number of charges that can be stored in the C-trap (∼1 million charges),28 and extremely high ion populations in the C-trap lead to space-charge effects,

34 which in turn deteriorate the SA.41 Based on the previous study on SA in FT-based mass spectrometers,4 a χ2 of less than 2.0 was chosen as the cutoff value for good spectral accuracy.

2 When comparing absolute monoisotopic ion abundances corresponding to χ < 2.0 in caffeineSigma,

MRFA, and ultramark 1421, we noticed that heavier molecules required higher absolute ion abundances for good spectral accuracy: absolute monoisotopic ion abundance of 1.9 × 104 is required to achieve high spectral accuracy in caffeineSigma, while absolute monoisotopic ion abundances of 2.0 × 104 and 4.6 × 104 are required for accurate measurement of isotopic distributions in MRFA and ultramark 1421, respectively. These estimated absolute ion abundances can be useful in untargeted metabolomics studies, specifically in MSI, where many compounds are analyzed simultaneously.

The identification list in untargeted MSI analyses can be shortened, sometimes down to one unique elemental composition, when absolute ion abundances are above-reported thresholds.

We propose a workflow for efficient elucidation of unknown elemental compositions relying on

MS1 spectra only (Figure 2.6A). First, the user generates a list with potential IDs using m/z for monoisotopic peak (±2.5 ppm). Next, relative abundances in the experimental isotopic distribution are used to estimate the number of atoms which in turn can help to eliminate IDs that do not match expected elemental compositions in the first list. When absolute ion abundances are above- reported minimum, estimation is highly accurate and thus chances of eliminating “correct” ID are very small. However, if absolute abundance is smaller than what we report in this work, the user may not be able to efficiently reduce the ID list because, as we have already mentioned previously, low-abundant species have a deteriorated isotopic distribution that cannot be used for accurate estimations of the number of atoms. We should also emphasize that the proposed workflow in

Figure 2.6A will not always work. For instance, if the generated ID list has only isomers, even the

2 Figure 2.5. Pearson χ distributions for (A) caffeineSigma, (B) MRFA, and (C) ultramark 1421 across eight AGC targets. Each data point represents a single transient scan.

36 highest SA will not help to distinguish those because Orbitrap-based mass spectrometers do not resolve isomeric peaks. Even though high MMA coupled with SA does not always result in identification of unique elemental compositions, MMA and SA can definitely help to narrow down an ID list, resulting in fewer molecules to consider for MS/MS analyses.

To better explain how reported thresholds for absolute ion abundances from direct infusion

experiments can be implemented in untargeted MSI analyses, we estimated the number of carbons and sulfurs in tissues with low and high absolute ion abundances of cholesterol C27H46O

(Figure 2.6B) and glutathione C10H17N3O6S (Figure 2.6C,D). For carbon and sulfur counting in Figure 2.6, we used atom percent values (best measurements) adopted from IUPAC.

Ion heat maps in Figure 2.6B–D were generated in MSiReader, free, open-source software for analyses of MSI files.42, 43 The SA tools that we used for atom counting were readily implemented using MSiReader’s programming interface. These tools will be incorporated in the next free release.43 When correlating carbon and sulfur counting with absolute ion abundances in healthy (low-abundant signal) and cancerous (high-abundant signal) tissues, two major points arise. First, Figures 2.6B–D show that carbon and sulfur counting is much more reliable in cancerous tissues with higher ion abundance than in healthy ones. This once again explains why knowing thresholds for absolute ion abundances is crucial when counting atoms. Second, Figure

2.6B confirms that for accurate carbon counting in a relatively small nonsulfur containing

3 13 compound, at least 7.9 × 10 of C1 ions are required whereas for accurate carbon (Figure 2.6C) and sulfur (Figure 2.6D) counting in a relatively small sulfur-containing compound, absolute ion

13 34 3 2 abundance of C1 and S1 ions has to be at least 1.1 × 10 and 6.6 × 10 , respectively.

The results of the studies presented here show that while the Orbitrap is not as precise as

IRMS for atomic analyses, it can be confidently used for determination of elemental compositions

Figure 2.6. (A) Proposed workflow for elucidation of unknown elemental compositions in untargeted MSI experiments. (B) Ion maps for cholesterol in healthy and cancerous hen ovarian tissue sections, showing increased abundance in cancerous tissue and thus more accurate carbon counting. Ion maps for glutathione in healthy and cancerous hen ovarian tissue sections, showing increased abundance in cancerous tissue and thus more accurate (C) carbon and (D) sulfur + + + − counting. Cholesterol [M – H2O + H ] and glutathione [M – H ] ions were generated in positive and negative ESI modes (IT = 110 ms), respectively, using 1 mM acetic acid in 50:50 MeOH:H2O as electrospray solvent. Note that atom percent values (e.g., 1.1078%), used for carbon and sulfur counting, were adopted from IUPAC.

38 of unknowns at optimum ion populations. We established thresholds for absolute ion abundances that are required to achieve high SA in molecules spanning a wide mass range. The high SA in tandem with high MMA are needed to identify unknown analytes observed in untargeted IR-

MALDESI MSI analyses. Current efforts are focused on implementing the screening thresholds in the image processing software MSiReader42, 43 for automated assessment of SA.

2.4 Conclusions

Elucidation of unknown elemental compositions requires not only accurately measured mass-to-charge ratios but also accurately measured relative abundances because they allow for the estimation of elemental compositions. In this work we showed that relative abundances measured in the Orbitrap ultimately depend on the number of ions injected into the mass analyzer. We also demonstrated that when absolute ion abundances are above the recommended thresholds, the Q

Exactive Plus can be used for accurate estimation of the number of carbons, nitrogens, and sulfurs in small (100 < MW (Da) < 400), medium (400 < MW (Da) < 900), and large (1000< MW (Da) <

1500) compounds. We used caffeineSigma, MRFA, and ultramark 1421 as representative targets for

13 the three different mass ranges. Relative amplitudes of the resolved C1 molecular isotope can predict the correct number of carbon atoms within the tolerance of less than ±0.5 carbons when

13 3 3 the absolute ion abundance of the C1 peak is at least 7.9 × 10 in caffeineSigma, 1.1 × 10 in

MRFA, and 1.5 × 104 in ultramark 1421. Similarly, the abundances of the resolved isotope peaks

15 34 of N1 and S1 enable the correct number of nitrogens and sulfurs within less than ±0.5 atoms

15 34 2 tolerance when absolute ion abundance of N1 and S1 peaks is 3.4 × 10 in caffeineSigma and 6.6

× 102 in MRFA, respectively.

Using Pearson χ2 distributions, we have also shown that as the molecule’s mass increases, more ions of that heavy molecule should be injected into the Orbitrap to accurately estimate the

39 number of carbons, nitrogens, and sulfurs. χ2 distributions suggest that for accurate estimation of elemental compositions in small (100 < MW (Da) < 400), medium (400 < MW (Da) < 900), and large (1000 < MW (Da) < 1500) compounds, absolute monoisotopic ion abundance in those compounds must be at least 1.9 × 104, 2.0 × 104, and 4.6 × 104, respectively.

2.5 Acknowledgments

The authors gratefully acknowledge the financial support received from the National

Institutes of Health (R01GM087964), the W. M. Keck Foundation, and North Carolina State

University. This work was supported in part by a grant from the National Institute of Justice: NIJ

2013-DN-BX-K007. The authors also thank United States Geological Survey (Reston Stable

Isotope Laboratory, Reston, VA) for measuring δ34S in provided MRFA samples.

2.6 References

1. Miura, D.; Tsuji, Y.; Takahashi, K.; Wariishi, H.; Saito, K. Anal. Chem. 2010, 82, 887−5891.

2. Hu, Q.; Noll, R. J.; Li, H.; Makarov, A.; Hardman, M.; Graham Cooks, R. J. Mass Spectrom. 2005, 40, 430−443.

3. Makarov, A.; Denisov, E.; Lange, O.; Horning, S. J. Am. Soc. Mass Spectrom. 2006, 17, 977−982.

4. Blake, S. L.; Walker, S. H.; Muddiman, D. C.; Hinks, D.; Beck, K. R. J. Am. Soc. Mass Spectrom. 2011, 22, 2269−2275.

5. Wang, Y.; Gu, M. Anal. Chem. 2010, 82, 7055−7062.

6. Kind, T.; Fiehn, O. BMC Bioinf. 2006, 7, 234.

7. Nazari, M.; Muddiman, D. C. Analyst 2016, 141, 595−605.

8. Weber, R. J.; Southam, A. D.; Sommer, U.; Viant, M. R. Anal. Chem. 2011, 83, 3737−3743.

9. Su, X.; Lu, W.; Rabinowitz, J. D. Anal. Chem. 2017, 89, 5940−5948.

10. Erve, J. C. L.; Gu, M.; Wang, Y.; DeMaio, W.; Talaat, R. E. J. Am. Soc. Mass Spectrom. 2009, 20, 2058−2069.

11. Kind, T.; Fiehn, O. BMC Bioinf. 2007, 8, 105.

12. Pluskal, T. s.; Uehara, T.; Yanagida, M. Anal. Chem. 2012, 84, 4396−4403.

13. Erve, J. C.; Gu, M.; Wang, Y.; DeMaio, W.; Talaat, R. E. J. Am. Soc. Mass Spectrom. 2009, 20, 2058−2069.

14. Xu, Y.; Heilier, J.-F.; Madalinski, G.; Genin, E.; Ezan, E.; Tabet, J.-C.; Junot, C. Anal. Chem. 2010, 82, 5490−5501.

15. March, R. E.; Stairs, R. A.; Stock, N. L. Int. J. Mass Spectrom. 2017, 415, 18−28.

16. Berglund, M.; Wieser, M. E. Pure Appl. Chem. 2011, 83, 397−410.

17. Makarov, A. Anal. Chem. 2000, 72, 1156−1162.

18. Hardman, M.; Makarov, A. A. Anal. Chem. 2003, 75, 1699−1705.

19. Makarov, A.; Denisov, E.; Kholomeev, A.; Balschun, W.; Lange, O.; Strupat, K.; Horning, S. Anal. Chem. 2006, 78, 2113−2120.

20. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712−1716.

21. Bokhart, M. T.; Muddiman, D. Analyst 2016, 141, 5236−5245.

22. Barry, J. A.; Robichaud, G.; Bokhart, M. T.; Thompson, C.; Sykes, C.; Kashuba, A. D.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2014, 25, 2038−2047.

23. Rosen, E. P.; Bokhart, M. T.; Nazari, M.; Muddiman, D. C. Anal. Chem. 2015, 87, 10483−10490.

24. Smith, C. A.; O’Maille, G.; Want, E. J.; Qin, C.; Trauger, S. A.; Brandon, T. R.; Custodio, D. E.; Abagyan, R.; Siuzdak, G. Ther. Drug Monit. 2005, 27, 747−751.

25. Wishart, D. S.; Tzur, D.; Knox, C.; Eisner, R.; Guo, A. C.; Young, N.; Cheng, D.; Jewell, K.; Arndt, D.; Sawhney, S.; et al. Nucleic Acids Res. 2007, 35, D521−D526.

26. Itano, W. M.; Bergquist, J. C.; Bollinger, J. J.; Wineland, D. J. Phys. Scr. 1995, 1995, 106.

27. Scheltema, R. A.; Hauschild, J.-P.; Lange, O.; Hornburg, D.; Denisov, E.; Damoc, E.; Kuehn, A.; Makarov, A.; Mann, M. Mol. Cell. Proteomics 2014, 13, 3698−3708.

28. Olsen, J. V.; de Godoy, L. M. F.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.; Lange, O.; Horning, S.; Mann, M. Mol. Cell. Proteomics 2005, 4, 2010−2021.

29. Kessner, D.; Chambers, M.; Burke, R.; Agus, D.; Mallick, P. Bioinformatics 2008, 24, 2534−2536.

30. Bantscheff, M.; Boesche, M.; Eberhard, D.; Matthieson, T.; Sweetman, G.; Kuster, B. Mol. Cell. Proteomics 2008, 7, 1702−1713.

31. Muccio, Z.; Jackson, G. P. Analyst 2009, 134, 213−222.

32. Carter, J.; Fry, B. Anal. Bioanal. Chem. 2013, 405, 2799−2814.

33. Santrock, J.; Studley, S. A.; Hayes, J. Anal. Chem. 1985, 57, 1444−1448.

34. Brenna, J. T.; Corso, T. N.; Tobias, H. J.; Caimi, R. J. Mass Spectrom. Rev. 1997, 16, 227−258.

35. Coplen, T. B.; Krouse, H. R. Nature 1998, 392, 32.

36. Kozhinov, A. Fourier transform mass spectrometry at the uncertainty principle limit for improved qualitative and quantitative molecular analyses. Thesis, EPFL, Lausanne, 2015.

37. Miladinovic, S. a. M.; Kozhinov, A. N.; Gorshkov, M. V.; Tsybin, Y. O. Anal. Chem. 2012, 84, 4042−4051.

38. Zubarev, R. A.; Makarov, A. Anal. Chem. 2013, 85 (11), 5288−5296.

39. Hu, Q.; Makarov, A. A.; Cooks, R. G.; Noll, R. J. J. Phys. Chem. A., 2006, 110, 2682−2689.

40. Gordon, E. F.; Muddiman, D. C. J. Mass Spectrom. 2001, 36, 195−203.

41. Gorshkov, M. V.; Good, D. M.; Lyutvinskiy, Y.; Yang, H.; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2010, 21, 1846−1851.

42. Robichaud, G.; Garrard, K. P.; Barry, J. A.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2013, 24, 718−721.

43. Bokhart, M. T.; Nazari, M.; Garrard, K. P.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2017, 1−9.

CHAPTER 3

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) and

Shotgun Provide Complementary Information about Lipidome Coverage

3.1 Introduction

Lipidomics is one of the youngest disciplines in the -omics field. The term ‘lipidomics’ was introduced in the early 2000s primarily due to the recognition of its importance and close relation to genomics, proteomics, and metabolomics.1,2 Lipidomics is a study of the entire collection of chemically distinct lipids in a cell, an organ, or a biological system.3 Lipids are hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion- based condensation of thioesters (fatty acids, polyketides, etc.) and/or by carbocation-based condensation of isoprene units (prenols, sterols, etc.).1,2,3,4,5,6 Lipids play many crucial roles in biological systems such as storing energy, signaling, and acting as structural components of cell membranes.4,5,7,8,9,10,11,12 Lipids are associated with many human diseases such as diabetes, obesity, stroke, cancer, psychiatric disorder, neurodegenerative diseases, neurological disorders, and infectious diseases among many others.1,3,7,13,14,15,16,17,18,19,20,21,22

Significant advancements and discoveries have been made in the field of lipidomics over the past two decades; however, the enormous structural diversity of lipids still remains a significant analytical challenge. Overall, lipids can be grouped into 8 categories based on their chemical structures and biological origins: sterols, glycerolipids, fatty acyls, sphingolipids, saccharolipids, prenols, glycerophospholipids, and polyketides.3,23 Each lipid category can be further broken down into main classes (over 550 lipid classes have been identified so far24) depending on the composition of acyl chains, the number and position of double bonds, stereochemistry, head group, and the linkage between backbone and its components.1,2,4,25,26 Due to the number and variety of

44 building blocks that lipids are made of, there are a significant number of isobaric and isomeric lipid species. For instance, the glycerophospholipid (GP) lipid category alone consists of approximately 180,000 theoretical glycerophospholipids2, illustrating the requirement for a powerful analytical tool to allow for their successful separation and subsequent identification.7

Nuclear magnetic resonance (NMR), fluorescence spectroscopy, high-performance liquid chromatography (HPLC), and microfluidic devices have advanced the field of lipidomics; however, advancements in mass spectrometry, including the development of ESI and MALDI, have tremendously accelerated the progress in the field of lipidomics.1,2,5,27,28 Direct infusion (i.e., shotgun) and LC-MS based approaches are the most frequently used analytical techniques in untargeted lipidomics. In shotgun, a crude lipid extract is diluted in ESI solvent and directly infused into a mass spectrometer without prior purification steps. One of the major advantages of shotgun is that molecular ions of all lipid categories can be displayed in a single MS1 spectrum which allows for a high-throughput characterization of the lipidome.29 Infusion at constant concentrations allows the optimization of instrumental parameters such as collision energies;1,15 and ionization of all lipid categories simultaneously makes analyses with different MS modes

(positive and negative) straightforward. In contrast, LC-MS is a more sensitive technique compared to shotgun because there is less ion suppression due to different elution times of low- and high-abundant lipids. LC-MS relies on a solvent gradient to resolve individual lipid species chromatographically; therefore, the chemical diversity of lipids requires careful selection of the solvent and column chemistry. The only major drawback of both shotgun and LC-MS is the lack of spatial information – one of the key parameters for understanding the physiological role of biomolecules.30

Mass spectrometry imaging (MSI) is an analytical strategy used for a localization of molecular features and their relative abundances in a sample. A typical MSI system consists of an ionization source and a mass spectrometer. The ionization source extracts and ionizes biomolecules from the sample surface and their mass-to-charge (m/z) ratios are recorded at each location. The ionization source dictates what molecular classes will be extracted and ionized from a sample and the quality of mass spectra depends on the mass analyzer. There is not a single ionization source that can extract and ionize all species present in a sample. However, almost all ionization sources used in MSI have been successfully employed for detection of lipids including

MALDI31, DESI32, SIMS33, pressurized liquid extraction surface analysis (LESA)34, direct analysis in real time (DART)35, rapid evaporative ionization mass spectrometry (REIMS)36, and

IR-MALDESI20. However, little is known about a range of lipid species that can be confidently detected by each MSI strategy. While a comprehensive review of lipidome coverage by each ionization source is beyond the scope of this study, the objective of this work is to reveal lipid species that can be confidently detected by IR-MALDESI and therefore reduce false discovery rates, given the complexity and diversity of lipids.

IR-MALDESI is a soft ionization source37 that has been used in MSI to monitor the spatial distributions of different molecules in biological38,39,40,41,42,43,44,45 and non-biological samples. 46,47

IR-MALDESI combines the properties of ESI and MALDI and has several distinctive characteristics: prior to analysis, the sample undergoes no chemical modifications48; ice is used as the only energy-absorbing matrix that facilitates desorption/ablation4950; ionization takes place at ambient pressure; and singly- and multiply-charged ions can be generated by the source50. The fundamentals and operation of the IR-MALDESI source have been described elsewhere51. Briefly, when imaging soft tissues, the sample is sliced in 5- to 200-µm-thick sections using a

46 cryomicrotome and thaw-mounted onto a glass slide, which is then placed onto a Peltier-cooled translation stage. After a thin ice layer is formed on top of the sample, a mid-IR laser pulse (2940 nm) is fired one spot at a time to ablate primarily neutral species by excitation of O-H stretching modes in the exogenous ice and endogenous water.52 These neutrals are ejected orthogonal to the surface and partition into charged ESI droplets. Next, electric field draws charged droplets into a mass spectrometer where they undergo desolvation and charge transfer in an ESI-like manner.52

And finally, the stage moves to the next location for analysis of adjacent spot.

When imaging biological samples using IR-MALDESI, we observe abundant lipid ions in both positive and negative ionization modes.20,48,53,54,55 We previously reported 399 and 727 unique lipids that were putatively annotated (based on MS1 only) in healthy and cancerous hen ovaries by searching accurate precursor (MS1) masses against METLIN and LIPID MAPS databases with a 5-ppm tolerance.20 However, we have not defined the lipidome fraction that can be detected via

IR-MALDESI imaging platform. To reveal a range of lipid classes that can be reliably observed in IR-MALDESI source, we compared lipidome coverage from a typical IR-MALDESI imaging experiment to that of a direct shotgun-type experiment because both approaches lack separation steps prior to ionization. It is worth noting that Nguyen et al.32 compared the depth of lipidome coverage in a nano-DESI to that of LC-MS/MS tissue extracts and nano-DESI, just like IR-

MALDESI, involves minimal to absolutely no sample preparation steps. Their findings show that fatty acyls, sphingolipids, phospholipids, and glycerolipids can be detected by nano-DESI. The same lipid classes were detected by IR-MALDESI.

3.2 Methods

3.2.1 Materials

ACS certified 2-methylbutane, OptimaTM LC/MS grade methanol and water were

47 purchased from Fisher Scientific (Pittsburgh, PA, USA). Chloroform with 100-200 ppm amylene as stabilizer (≥99.5%), sodium chloride, and acetic acid were purchased from Sigma-Aldrich (St

Louis, MO, USA). Nitrogen gas was obtained from Arc3 Gases (Raleigh, NC, USA) and plain microscope slides were purchased from VWR (Radnor, PA, USA).

3.2.2 Preparation of Rat Liver for Shotgun and IR-MALDESI MSI

Liver from a healthy adult Sprague-Dawley rat was used for both IR-MALDESI and shotgun analyses. A 100-µm-thick section from the center of the liver was used for IR-MALDESI

MSI, and the remaining liver tissue was used for shotgun analysis. Liver was sliced on a Leica CM

1950 cryomicrotome (Buffalo Grove, IL, USA) using a high-profile coated microtome blade from

VWR and (Batavia, IL, USA). Chamber temperature during sectioning was maintained at -15 °C.

3.2.3 Direct Infusion

Prior to shotgun analysis, lipids were extracted from a fresh rat liver using a modified Folch method56. Briefly, rat liver slices were ground in liquid nitrogen and 80.30 mg of frozen rat liver was homogenized with 1526 µL of 2:1 (v/v) chloroform:methanol solution to have a final volume roughly 20 times the volume of the tissue. The mixture was homogenized in a cell disruptor for 5 minutes (to release lipids from cells) and centrifuged for 5 minutes (2000 rpm, 4 °C) to remove tissue debris. After centrifugation, solvent was transferred into a new Eppendorf tube leaving tissue behind. The homogenate was washed with 0.1% sodium chloride (20% of the homogenate volume). After a 5-minute centrifugation, the organic layer with dissolved lipids was removed and dried under nitrogen stream. Dried lipids were re-suspended in 200 µL of 0.1% acetic acid in 2:1

(v/v) chloroform:methanol and directly infused into a Q Exactive Plus mass spectrometer (Thermo

Fisher Scientific, Bremen, Germany) for analysis. Sample was introduced into a mass spectrometer using a Fusion 101 digital syringe pump (Thermo Fisher Scientific, Bremen, Germany) equipped

48 with a HamiltonTM GastightTM syringe (Thermo Fisher Scientific, Pittsburgh, PA, USA) at a flow rate of 2 µL/min. Spray voltage was set to 4.00 kV, MS inlet capillary temperature was 315

°C, and S-lens RF level was set to 50%.

3.2.4 IR-MALDESI MSI

A 100-µm-thick section from a rat liver was mounted onto the translation stage to collect mass spectra from the liver slice in 20 rows (x-direction) and 30 lines (y-direction) with a spot-to- spot spacing of 150 µm. Two mid-IR laser pulses from OpoletteTM SE 2731/3034 tunable laser

(Opotek, Carlsbad, CA, USA) with a frequency of 20 Hz and pulse width of 5-7 ns were used to desorb neutrals from each voxel on the rat liver. Incident energy of approximately 1 mJ per pulse was measured using a Nova 2 pyroelectric detector (Ophir, Jerusalem, Israel). ESI solvent for positive ESI mode was 0.2% formic acid and 1 mM acetic acid20 in 50:50 (v/v) methanol:water for negative mode. ESI flow rate was set to 2 µL/min, spray voltage was 4.00 kV, MS inlet capillary temperature was 315 °C, and S-lens RF level was set to 50%.

3.2.5 Data Collection and Analysis

All mass spectrometry measurements were performed on a Q Exactive Plus (Thermo Fisher

Scientific, Bremen, Germany) mass spectrometer. Data were collected across low (m/z 150-600) and high (m/z 500-2000) mass ranges in both polarities. Data-dependent acquisition (DDA) mode was used as a rapid data collection technique for both shotgun and IR-MALDESI analyses. DDA proceeds in cycles, typically consisting of a full MS1 scan followed by consecutive MS/MS scans where TopN of the precursor ions are selected for fragmentation in order of abundance. The Q

Exactive Plus software allows up to 20 MS/MS spectra per DDA cycle. Following fragmentation, precursor ions are appended to a dynamic exclusion list for a specified amount of time to avoid fragmentation of the same ions in the next DDA cycles. In the work presented here, both shotgun

49 and IR-MALDESI analyses had method and dynamic exclusion set to 6 minutes to ensure that precursor ions were fragmented only once. Prior to analysis of rat liver samples, we directly infused solvents used for IR-MALDESI (0.2% formic acid and 1 mM acetic acid in 50:50 (v/v) methanol:water) and shotgun (0.1% acetic acid in 2:1 (v/v) chloroform:methanol) to record 100 most abundant background ions (Table B.1). These background ions were included in the DDA exclusion list. DDA parameters in both shotgun and IR-MALDESI were the same: resolving power of 140,000fwhm at m/z 200, 1 microscan, isolation window of 1.3 m/z, normalized collision energy of 20%. Automatic gain control (AGC) target and IT for MS1 scans in the DDA method were equal to 3×106 and 75 ms. However, IR-MALDESI is a pulsed ionization source where species from the sample are ionized only after the laser ablation event.53 To synchronize laser ablation with ion collection, AGC target in IR-MALDESI is always disabled and IT of 75 ms is the only parameter that controls ion population in the C-trap.57 In shotgun, on the other hand, AGC is always on, because there is no delay in accumulation of analyte ions.

Lipids were automatically identified by searching their accurate masses for precursor and product ions using LipidSearch 4.1.16 (Thermo Fisher Scientific, Bremen, Germany). LipidSearch contains over 1.7 million lipid ions and their predicted fragment ions. Lipid classes that were annotated by LipidSearch are listed in the (Table B.2). The comprehensive identification algorithm for product-ion scans discriminates each lipid by matching the observed fragmentation pattern with the predicted ones stored in the LipidSearch database. Mass tolerance for precursor and product ions was set to 5 and 10 ppm, based on recommended settings for the instrument and type of analysis. LipidSearch reports not only lipid IDs and classes but also the quality metrics such as m-score, grade, and mass measurement accuracy (MMA). The m-score is calculated based on the number of matches with the product ion: the higher the m-score the more accurate is

50 identification. Grade indicates accuracy of identification: A is the highest and D is the lowest grade. In grade A, headgroup and all fatty acid chains are completely identified. In grade B, headgroup and some fatty-acid-derived product ions are annotated. In grade C, either headgroup or fatty-acid-derived product ions are detected. And finally, in grade D, the lipid identity is inferred only from the presence of peaks originating from other non-specific reactions (i.e., water loss). In the work presented here, m-score threshold was set to 5 and ID quality filters were set to include lipids with grades A, B, and C. Lipid ions that had the same ID but different adducts (e.g., [lipid

+ + + + X +H ] and [lipid X +H -H2O] ) were kept in the list. Table with all annotated lipid ions is shown in the (TableB.3).

3.3 Results and Discussion

3.3.1 Lipidome Coverage via IR-MALDESI and Shotgun

The goal of this work was not to maximize the total number of lipids but to determine the extent of overlap and complementarity shotgun and IR-MALDESI techniques have. We aimed to investigate lipid ions, IDs, classes, and categories that can be detected via IR-MALDESI source coupled with a Q Exactive Plus mass spectrometer. To generate a list with lipids that can be successfully extracted and ionized via IR-MALDESI source, we analyzed the same rat liver via shotgun and IR-MALDESI across a wide mass range, in both positive and negative ionization modes. Lipidome coverage in IR-MALDESI was compared to lipidome coverage in shotgun because both techniques lack separation steps prior to ionization; and therefore, shotgun mimics

IR-MALDESI as an LC-MS approach reduces ionization suppression not allowing for a direct and fair comparison.

To automatically annotate peaks representing lipids and learn more about the quality of lipidome coverage in shotgun and IR-MALDESI, precursor and their fragment ions were annotated

51 using LipidSearchTM software. The list with all precursor and product ions annotated in

LipidSearch is shown in the (Figure B.3). In this work we discuss only lipids that were annotated based on MS2.

In a typical untargeted IR-MALDESI analysis, collecting MS/MS for every feature from every voxel is challenging. Therefore, in untargeted IR-MALDESI analyses we rely solely on mass and spectral accuracy to estimate elemental composition from isotopic distributions.58 Figure 3.1 shows representative MS2 (Figures 3.1A, 3.1C) and MS1 (Figures 3.1B, 3.1D) spectra for FA

(20:4) detected via shotgun and IR-MALDESI. When MS/MS for a specific precursor m/z is not available, we can use the isotopic distribution (Figures 3.1B, 3.1D) to estimate the number of

58 13 elements in an unknown. For example, knowing the relative abundance of C1 isotopologue

(molecule with one 13C), we can estimated the number of carbons that equals to the abundance

13 13 58,59 ratio of C1 isotopologue to that of C isotope in nature (approximately 1.11%). Based on these calculations, we estimated 19 carbons from shotgun and 20 carbons from IR-MALDESI experiments.

We hypothesize that IR-MALDESI and shotgun will not provide identical information about the lipidome because of the extraction bias and the amount of material used for each analysis.

In IR-MALDESI MSI, lipids are extracted from a thin biological section using the energy from a mid-IR laser and in shotgun lipids are extracted using a liquid solid extraction. Moreover, in IR-

MALDESI experiments lipids are detected at native concentrations from minute amounts of material whereas the concentration of lipids in a shotgun-type experiment is several orders of magnitude higher. In fact, the total amount (mass) of tissue typically used for shotgun lipidomics is 8 orders of magnitude higher compared to the amount rat liver tissue used for IR-MALDESI imaging. Given that section thickness, ablation spot, and liver density were 100 µm, 100×100 µm,

Figure 3.1. MS2 (A, C) and MS1 (B, D) spectra for FA (20:4) detected in shotgun and IR- MALDESI analyses. Fragmentation patterns of precursor ion was searched against METLIN database. and 1 g/cm3 µm, we estimated that 80.0×10-8 mg of rat liver was ablated from each voxel in IR-

MALDESI compared to 80.3 mg of rat liver used for Folch extraction.

Representative MS1 spectra collected across low (m/z 150-600) and high (m/z 500-2000) mass ranges in both positive and negative ion modes are shown in (Figure 3.2). Note that labels represent lipid classes that were confidently annotated in LipidSearch based on MS2 patterns. The following key observations can be highlighted from (Figure 3.2): zymosterol ester and small fatty acyls were detected only via IR-MALDESI across low mass range in positive and negative modes,

53 respectively; and cardiolipins were detected only via shotgun in negative mode across high mass range. Overall, the same 5 lipid categories were detected by both shotgun and IR-MALDESI

Figure 3.2. Representative MS1 spectra collected by shotgun and IR-MALDESI. Ion abundances were normalized to the most abundant peak in each spectrum to allow for easer comparison. Peaks were labeled based on confident (MS/MS) annotations from LipidSearch.

54 techniques: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterols. It is worth noting that LipidSearch annotated no lipids within m/z 1500-1700 (positive mode) both in shotgun and IR-MALDESI. However, METASPACE60 putatively annotated cardiolipin [M+Na+]+ ion at m/z 1568.1492 from shotgun MS1 data. Putative METASPACE IDs are listed in Table B.4.

Figure 3.3 shows that 70 and 341 unique lipid ions (Figure 3.3A), 53 and 296 lipid IDs

(Figure 3.3B), 1 and 19 lipid classes (Figure 3.3C) were detected exclusively by IR-MALDESI and shotgun, individually; whereas 11 lipid ions, 24 lipid IDs, and 11 classes were detected by both methods. Since lipidome coverage in shotgun-type analyses has already been investigated by others61,62,63, the motivation of this work was to characterize lipids that can be detected only via

IR-MALDESI as well as via both IR-MALDESI and shotgun. Seventy (70) unique lipid ions detected only via IR-MALDESI (Figure 3.3A) represent the following lipid classes: diglyceride

(DG), dimethylphosphatidylethanolamine (dMePE), fatty acid (FA), lysophosphatidylcholine

(LPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylethanol (PEt), phosphatidylglycerol (PG), phosphatidylmethanol (PMe), sphingomyelin (SM), triglyceride (TG), and zymosterol (ZyE). Eleven (11) lipid ions that were detected both in IR-MALDESI and shotgun represent FA, LPC, PC, and PE. Fifty three (53) unique lipid IDs (Figure 3.3B) that were identified only via IR-MALDESI represent the following lipid classes: DG, dMePE, FA, LPC, PC, PEt, PG,

PMe, SM, TG, and ZyE; and 24 lipid IDs that were annotated from both IR-MALDESI and shotgun data represent the following lipid classes: DG, dMePE, FA, LPC, PC, PE, and TG. Finally, lipid classes detected in shotgun and IR-MALDESI, almost fully overlap (Figure 3.3C): 1 lipid class (ZyE) was detected only in IR-MALDESI, 19 lipid classes were detected only in shotgun,

55 and 11 lipid classes (PE, PEt, PG, PMe, SM, TG, DG, dMePE, FA, LPC, and PC) were identified by both methods.

Figure 3.3. Venn diagram showing the number of (A) lipid ions, (B) lipid IDs, and (C) lipid classes detected via IR-MALDESI and shotgun analyses. Note that annotations are based on MS2.

It is worth noting that zymosterol, precursor of cholesterol, was previously detected via IR-

+ + 107 + + 54 MALDESI as [M-H2O+H ] at m/z 367.3365 and [M+ Ag ] at m/z 491.2457. However, in this work only zymosterol [M+H+]+ ion was annotated because LipidSearchTM version 4.1.16 does not

+ + + + assign IDs to dehydrated molecular ions, although both [M+H ] and [M-H2O+H ] zymosterols

+ + + + were detected via IR-MALDESI, and neither [M+H ] nor [M-H2O+H ] zymosterol ions were detected in shotgun. This can be explained by poor ionization efficiency of free cholesterols that usually require derivatization to improve ionization in ESI.

To better understand the extraction and ionization of lipids in IR-MALDESI source, we compared lipid ions in the overlapping region of the Venn diagram to monitor any significant differences between ion abundance, and grade (Figure 3.4A), as well as MMA and m-score

(Figure 3.4B). As expected, abundance ratios in shotgun are higher compared to abundance ratios in IR-MALDESI because in the former we worked with significantly more concentrated samples.

The only exception is LPC(16:1p)+H+ ion whose abundance ratio in IR-MALDESI is slightly

56 higher compared to abundance ratio in shotgun. Figure 3.4A also shows that grade of PE lipids in shotgun is slightly higher compared to grade of PE lipids in IR-MALDESI. However, the rest of

Figure 3.4. (A) Abundance ratio (left y-axis) and grade (right y-axis) distributions for lipid ions (x-axis) detected in shotgun (blue bars) and IR-MALDESI (orange bars). Abundance ratios were calculated by dividing abundance of a lipid, detected in shotgun or IR-MALDESI, by the sum of abundances from both techniques: 퐴 퐴 푠ℎ표푡𝑔푢푛 × 100% or 퐼푅−푀퐴퐿퐷퐸푆퐼 × 100% 퐴푠ℎ표푡𝑔푢푛 +퐴퐼푅−푀퐴퐿퐷퐸푆퐼 퐴푠ℎ표푡𝑔푢푛 +퐴퐼푅−푀퐴퐿퐷퐸푆퐼 (B) MMA (left y-axis) and m-score (right y-axis) distributions for lipids ions (x-axis) that were annotated both in shotgun and IR-MALDESI.

57 the ‘overlapping’ lipid ions have the same grade.

In summary, Figures 3.4A and 3.4B show that ion abundances are not directly correlated to grades or m-scores. For example, in shotgun, PC(16:0_16:1)+H+ has abundance ratio of 83% and is a grade C lipid; on the contrary, LPC(16:1p)+H+ has abundance ratio of 29% and is a grade

B lipid. Similarly, in IR-MALDESI, LPC(16:0)+H+ has abundance ratio of 5% and m-score of 50; however, LPC(16:1p)+H+ has abundance ratio of 71% and m-score of 20. Histograms showing the distribution of grades, MMAs, and m-score for all lipid ions detected via shotgun and IR-

MALDESI are shown in the (Figure B.1). Briefly, the smallest number of ions were sorted under grade A, followed by grade B and C that both have a comparable number of detected ions. MMA in both techniques is spread between -5 and 5 ppm; and m-scores span from 10 to 200 with IR-

MALDESI having overall lower m-score compared to shotgun.

To investigate what lipid IDs were identified in each class and in each ionization method

(IR-MALDESI and shotgun), we generated a histogram (Figure 3.5) showing the number of unique lipid IDs that were detected only via IR-MALDESI or only via shotgun. Note that Figure

3.5 does not represent lipid IDs that were detected both via IR-MALDESI and shotgun. For example, PE lipids were identified both in shotgun and IR-MALDESI; however, only 16 PE lipids that are unique to shotgun are represented in the Figure 3.5. The remaining ‘overlapping’ lipid classes comprise lipids that are unique to only IR-MALDESI or only shotgun. Figure 3.5 shows that the number of unique IDs in Folch extraction exceeds that of IR-MALDESI with the exception to DG and TG. This suggests that IR-MALDESI extracts glycerides better than shotgun approach.

10 and 1 unique DG lipids were detected via IR-MALDESI and shotgun, respectively; and 16 unique TG lipid classes were detected both via IR-MALDESI and shotgun analyses.

From the Venn diagram (Figure 3.3) we learned that out of all lipid ions annotated in IR-

Figure 3.5. The number of unique lipid IDs that were annotated only via shotgun or only via IR- MALDESI. Note that y-axis on this histogram shows only unique lipid IDs.

MALDESI and shotgun, 80.8% of all lipid ions were annotated only from shotgun data. Is it possible that these ions can still be found in the imaging raw data even though they were too low abundant to be selected for fragmentation in DDA? To answer this question, we searched for all lipid ions, annotated exclusively via shotgun, against all IR-MALDESI data (Figure 3.6A).

Specifically, we were looking for the masses of known precursor ions in all survey MS1 scans.

During the search, peaks with signal-to-noise ratio less than 4 were filtered out.64 Next, we did the same manipulation using the peaks annotated solely via IR-MALDESI; these peaks were then searched against shotgun raw data. (Figure 3.6B). Out of 352 lipid ions annotated via shotgun only, 89 (25%) lipid ions were found in IR-MALDESI raw data (Figure 3.6A). Similarly, out of

81 lipid ions identified via IR-MALDESI only, 53 (65%) lipid ions were found in shotgun raw data (Figure 3.6B). There are two reasons for why some lipids were not selected for DDA

59 fragmentation in both shotgun and IR-MALDESI. First, some ions were too low abundant to be selected for fragmentation in DDA method. Second, majority of precursor ions were not captured in every MS1 scan as shown in the (Figure B.2). We attribute the lack of abundant precursor ions in every voxel to the heterogeneous nature of the rat liver slice, matrix affect, and analytical variability. Therefore, there is a possibility that in DDA method some abundant peaks were not selected for MS2 at all.

Figure 3.6. The number of lipid ions detected only via shotgun (light blue), only via IR-MALDESI (orange) as well as the number of lipid ions that can be recovered via shotgun (dark blue) and IR-MALDESI (red). To ensure that in developed DDA method no lipid ions were missed in IR- MALDESI and shotgun, (A) m/z of lipid ions that were detected via shotgun only, were searched against IR-MALDESI raw data; (B) similarly, m/z of lipid ions that were detected via IR- MALDESI only, were searched against shotgun raw data. Grade D lipids and ions duplicates are not shown on these Venn diagrams.

3.4 Conclusions

IR-MALDESI MSI can be used for detection of a range of lipid classes that are not effectively detected in a shotgun-type experiment. Overall, the total of 422 lipid ions were confidently (MS2) annotated from a rat liver, precursors for 159 of which were detected with IR-

MALDESI exclusively. Glycoglycerolipids were detected in shotgun while neutral lipids, phospholipids, fatty acids, and sphingolipids were detected both in shotgun and IR-MALDESI.

There is no lipid category that can be detected solely by one technique: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids were detected both in shotgun and IR-

MALDESI. Our results suggest that lipidome coverage is directly dependent on sample quantity: approximately 5 times more lipid ions were detected in shotgun compared to IR-MALDESI primarily because the total amount of tissue used in shotgun was 8 orders of magnitude higher compared to IR-MALDESI. The positive and negative biases inherent to IR-MALDESI will be considered when designing experiments and evaluating untargeted lipidomics data.

3.5 Acknowledgements

The authors gratefully acknowledge the financial support received from National Institute of Health (R01GM087964). This work was performed in part by the Molecular Education, Technology and Research Innovation Center (METRIC) at NC State University, which is supported by North Carolina.

3.6 References

1. Han, X.: Lipidomics: Comprehensive Mass Spectrometry of Lipids. (2016)

2. Blanksby, S.J., Mitchell, T.W.: Advances in Mass Spectrometry for Lipidomics. Annu. Rev. Anal. Chem. (2010). https://doi.org/10.1146/annurev.anchem.111808.073705

3. Fahy, E., Subramaniam, S., Brown, H.A., Glass, C.K., Merrill, A.H., Murphy, R.C., Raetz, C.R.H., Russell, D.W., Seyama, Y., Shaw, W., Shimizu, T., Spener, F., van Meer, G., VanNieuwenhze, M.S., White, S.H., Witztum, J.L., Dennis, E.A.: A comprehensive classification system for lipids. Eur. J. Lipid Sci. Technol. (2005). https://doi.org/10.1002/ejlt.200405001

4. Ryan, E., Reid, G.E.: Chemical Derivatization and Ultrahigh Resolution and Accurate Mass Spectrometry Strategies for “shotgun” Lipidome Analysis. Acc. Chem. Res. (2016). https://doi.org/10.1021/acs.accounts.6b00030

5. Wenk, M.R.: The emerging field of lipidomics (vol 4, pg 594, 2005). Nat. Rev. DRUG Discov. (2005). https://doi.org/10.1038/nrd1776

6. Bou Khalil, M., Hou, W., Zhou, H., Elisma, F., Swayne, L.A., Blanchard, A.P., Yao, Z., Bennett, S.A.L., Figeys, D.: Lipidomics era: Accomplishments and challenges. Mass Spectrom. Rev. (2010). https://doi.org/10.1002/mas.20294

7. Blaženović, I., Shen, T., Mehta, S.S., Kind, T., Ji, J., Piparo, M., Cacciola, F., Mondello, L., Fiehn, O.: Increasing Compound Identification Rates in Untargeted Lipidomics Research with Liquid Chromatography Drift Time-Ion Mobility Mass Spectrometry. Anal. Chem. (2018). https://doi.org/10.1021/acs.analchem.8b01527

8. Wenk, M.R.: Lipidomics: New tools and applications, (2010)

9. Alex Brown, H.: Lipidomics: When apocrypha becomes canonical, (2012)

10. Shevchenko, A., Simons, K.: Lipidomics: Coming to grips with lipid diversity, (2010)

11. Coskun, Ü., Simons, K.: Cell membranes: The lipid perspective, (2011)

12. Klose, C., Surma, M.A., Simons, K.: Organellar lipidomics-background and perspectives, (2013)

13. Gorden, D.L., Myers, D.S., Ivanova, P.T., Fahy, E., Maurya, M.R., Gupta, S., Min, J., Spann, N.J., McDonald, J.G., Kelly, S.L., Duan, J., Sullards, M.C., Leiker, T.J., Barkley, R.M., Quehenberger, O., Armando, A.M., Milne, S.B., Mathews, T.P., Armstrong, M.D., Li, C., Melvin, W. V., Clements, R.H., Washington, M.K., Mendonsa, A.M., Witztum, J.L., Guan, Z., Glass, C.K., Murphy, R.C., Dennis, E.A., Merrill, A.H., Russell, D.W., Subramaniam, S., Brown, H.A.: Biomarkers of NAFLD progression: A lipidomics approach to an epidemic 1. J. Lipid Res. (2015). https://doi.org/10.1194/jlr.P056002

14. Sa, R., Zhang, W., Ge, J., Wei, X., Zhou, Y., Landzberg, D.R., Wang, Z., Han, X., Chen, L., Yin, H.: Discovering a critical transition state from nonalcoholic hepatosteatosis to nonalcoholic steatohepatitis by lipidomics and dynamical network biomarkers. J. Mol. Cell Biol. (2016). https://doi.org/10.1093/jmcb/mjw016

15. Fhaner, C.J., Liu, S., Ji, H., Simpson, R.J., Reid, G.E.: Comprehensive lipidome profiling of isogenic primary and metastatic colon adenocarcinoma cell lines. Anal. Chem. (2012). https://doi.org/10.1021/ac302154g

16. Tania, M., Khan, M.A., Song, Y.: Association of lipid metabolism with ovarian cancer, (2010)

17. Roy, D., Mondal, S., Wang, C., He, X., Khurana, A., Giri, S., Hoffmann, R., Jung, D.-B., Kim, S.H., Chini, E.N., Periera, J., Folmes, C.D., Mariani, A., Dowdy, S.C., Bakkum- Gamez, J.N., Riska, S.M., Oberg, A.L., Karoly, E.D., Bell, L.N., Chien, J., Shridhar, V.: Loss of HSulf-1 promotes altered lipid metabolism in ovarian cancer. Cancer Metab. (2014). https://doi.org/10.1186/2049-3002-2-13

18. Iorio, E., Mezzanzanica, D., Alberti, P., Spadaro, F., Ramoni, C., D’Ascenzo, S., Millimaggi, D., Pavan, A., Dolo, V., Canevari, S., Podo, F.: Alterations of Choline Phospholipid Metabolism in Ovarian Tumor Progression. Cancer Res. 65, 9369–9376 (2005). https://doi.org/10.1158/0008-5472.CAN-05-1146

19. Santos, C.R., Schulze, A.: Lipid metabolism in cancer, (2012)

20. Nazari, M., Muddiman, D.C.: Polarity switching mass spectrometry imaging of healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Analyst. (2016). https://doi.org/10.1039/c5an01513h

21. Hall, Z., Chu, Y., Griffin, J.L.: Liquid Extraction Surface Analysis Mass Spectrometry Method for Identifying the Presence and Severity of Nonalcoholic Fatty Liver Disease. Anal. Chem. (2017). https://doi.org/10.1021/acs.analchem.7b01097

22. Miyamura, N., Nakamura, T., Goto-Inoue, N., Zaima, N., Hayasaka, T., Yamasaki, T., Terai, S., Sakaida, I., Setou, M., Nishina, H.: Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver. Biochem. Biophys. Res. Commun. (2011). https://doi.org/10.1016/j.bbrc.2011.03.133

23. Pengcheng Fan, M.L.: Lipidomics in Health and Diseases - Beyond the Analysis of Lipids. J. Glycomics Lipidomics. 05, (2015). https://doi.org/10.4172/2153-0637.1000126

24. Papan, C., Penkov, S., Herzog, R., Thiele, C., Kurzchalia, T., Shevchenko, A.: Systematic screening for novel lipids by shotgun lipidomics. Anal. Chem. (2014). https://doi.org/10.1021/ac404083u

25. Ekroos, K.: Lipidomics: Technologies and Applications. (2012)

26. Stâhlman, M., Borén, J., Ekroos, K.: High-Throughput Molecular Lipidomics. In: Lipidomics: Technologies and Applications (2012)

27. Brown, H.A., Murphy, R.C.: Working towards an exegesis for lipids in biology, (2009)

28. Van Meer, G.: Cellular lipidomics. EMBO J. (2005). https://doi.org/10.1038/sj.emboj.7600798

29. Han, X., Gross, R.W.: Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: A bridge to lipidomics, (2003)

30. Schwamborn, K., Kriegsmann, M., Weichert, W.: MALDI imaging mass spectrometry — From bench to bedside, (2017)

31. Naito, H.K., David, J.A.: Laboratory considerations: determination of cholesterol, triglyceride, phospholipid, and other lipids in blood and tissues., (1984)

32. Nguyen, S.N., Kyle, J.E., Dautel, S.E., Sontag, R., Luders, T., Corley, R., Ansong, C., Carson, J., Laskin, J.: Lipid Coverage in Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Mouse Lung Tissues. Anal. Chem. (2019). https://doi.org/10.1021/acs.analchem.9b02045

33. Sastry, P.S., Kates, M.: Hydrolysis of Monogalactosyl and Digalactosyl Diglycerides by Specific Enzymes in Runner-Bean Leaves. Biochemistry. (1964). https://doi.org/10.1021/bi00897a016

34. Almeida, R., Berzina, Z., Arnspang, E.C., Baumgart, J., Vogt, J., Nitsch, R., Ejsing, C.S.: Quantitative spatial analysis of the mouse brain lipidome by pressurized liquid extraction surface analysis. Anal. Chem. (2015). https://doi.org/10.1021/ac503627z

35. Cody, R.B., McAlpin, C.R., Cox, C.R., Jensen, K.R., Voorhees, K.J.: Identification of bacteria by fatty acid profiling with direct analysis in real time mass spectrometry. Rapid Commun. Mass Spectrom. (2015). https://doi.org/10.1002/rcm.7309

36. Strittmatter, N., Lovrics, A., Sessler, J., McKenzie, J.S., Bodai, Z., Doria, M.L., Kucsma, N., Szakacs, G., Takats, Z.: Shotgun Lipidomic Profiling of the NCI60 Cell Line Panel Using Rapid Evaporative Ionization Mass Spectrometry. Anal. Chem. (2016). https://doi.org/10.1021/acs.analchem.6b00187

37. Tu, A., Muddiman, D.C.: Internal Energy Deposition in Infrared Matrix-Assisted Laser Desorption Electrospray Ionization With and Without the Use of Ice as a Matrix. J. Am. Soc. Mass Spectrom. (2019). https://doi.org/10.1007/s13361-019-02323-2

38. Barry, J.A., Robichaud, G., Bokhart, M.T., Thompson, C., Sykes, C., Kashuba, A.D.M., Muddiman, D.C.: Mapping antiretroviral drugs in tissue by IR-MALDESI MSI coupled to the Q Exactive and comparison with LC-MS/MS SRM assay. J. Am. Soc. Mass Spectrom. (2014). https://doi.org/10.1007/s13361-014-0884-1

39. Bokhart, M.T., Rosen, E., Thompson, C., Sykes, C., Kashuba, A.D.M., Muddiman, D.C.: Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization. Anal. Bioanal. Chem. (2015). https://doi.org/10.1007/s00216-014-8220-y

40. Ekelöf, M., McMurtrie, E.K., Nazari, M., Johanningsmeier, S.D., Muddiman, D.C.: Direct Analysis of Triterpenes from High-Salt Fermented Cucumbers Using Infrared Matrix- Assisted Laser Desorption Electrospray Ionization (IR-MALDESI). J. Am. Soc. Mass Spectrom. (2017). https://doi.org/10.1007/s13361-016-1541-7

41. Thompson, C.G., Bokhart, M.T., Sykes, C., Adamson, L., Fedoriw, Y., Luciw, P.A., Muddiman, D.C., Kashuba, A.D.M., Rosen, E.P.: Mass spectrometry imaging reveals heterogeneous efavirenz distribution within putative HIV reservoirs. Antimicrob. Agents Chemother. (2015). https://doi.org/10.1128/AAC.04952-14

42. Nazari, M., Malico, A.A., Ekelöf, M., Lund, S., Williams, G.J., Muddiman, D.C.: Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI. Anal. Bioanal. Chem. (2018). https://doi.org/10.1007/s00216-017-0570-9

43. Nazari, M., Bokhart, M.T., Loziuk, P.L., Muddiman, D.C.: Quantitative mass spectrometry imaging of glutathione in healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Analyst. (2018). https://doi.org/10.1039/c7an01828b

44. Thompson, C., Rosen, E., Bokhart, M., Prince, H., Sykes, C., Muddiman, D.C., Kashuba, A.D.M.: Mass Spectrometry Imaging of Hair Strands Allows for Evaluation of Long Term Antiretroviral Adherence. AIDS Res. Hum. Retroviruses. (2014). https://doi.org/10.1089/aid.2014.5126a.abstract

45. Rosen, E.P., Thompson, C.G., Bokhart, M.T., Prince, H.M.A., Sykes, C., Muddiman, D.C., Kashuba, A.D.M.: Analysis of Antiretrovirals in Single Hair Strands for Evaluation of Drug Adherence with Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging. Anal. Chem. (2016). https://doi.org/10.1021/acs.analchem.5b03794

46. Cochran, K.H., Barry, J.A., Robichaud, G., Muddiman, D.C.: Analysis of trace fibers by IR-MALDESI imaging coupled with high resolving power MS. Anal. Bioanal. Chem. (2015). https://doi.org/10.1007/s00216-014-8042-y

47. Cochran, K.H., Barry, J.A., Muddiman, D.C., Hinks, D.: Direct analysis of textile fabrics and dyes using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry. Anal. Chem. (2013). https://doi.org/10.1021/ac302519n

48. Nazari, M., Bokhart, M.T., Muddiman, D.C.: Whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). J. Vis. Exp. (2016). https://doi.org/10.3791/53942

49. Robichaud, G., Barry, J.A., Muddiman, D.C.: IR-MALDESI mass spectrometry imaging

of biological tissue sections using ice as a matrix. J. Am. Soc. Mass Spectrom. (2014). https://doi.org/10.1007/s13361-013-0787-6

50. Sampson, J.S., Hawkridge, A.M., Muddiman, D.C.: Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. (2006). https://doi.org/10.1016/j.jasms.2006.08.003

51. Goldschlager, T., Abdelkader, A., Kerr, J., Boundy, I., Jenkin, G.: Undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis. J. Vis. Exp. (2010). https://doi.org/10.3791/1707

52. Dixon, R.B., Muddiman, D.C.: Study of the ionization mechanism in hybrid laser based desorption techniques. Analyst. (2010). https://doi.org/10.1039/b926422a

53. Rosen, E.P., Bokhart, M.T., Nazari, M., Muddiman, D.C.: Influence of C-Trap Ion Accumulation Time on the Detectability of Analytes in IR-MALDESI MSI. Anal. Chem. (2015). https://doi.org/10.1021/acs.analchem.5b02641

54. Loziuk, P., Meier, F., Johnson, C., Ghashghaei, H.T., Muddiman, D.C.: TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR- MALDESI imaging of lipids and LC-MS/MS label-free proteomics. Anal. Bioanal. Chem. (2016). https://doi.org/10.1007/s00216-016-9421-3

55. Rosen, E.P., Bokhart, M.T., Ghashghaei, H.T., Muddiman, D.C.: Influence of desorption conditions on analyte sensitivity and internal energy in discrete tissue or whole body imaging by IR-MALDESI. J. Am. Soc. Mass Spectrom. (2015). https://doi.org/10.1007/s13361-015-1114-1

56. FOLCH, J., LEES, M., SLOANE STANLEY, G.H.: A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. (1957). https://doi.org/10.3989/scimar.2005.69n187

57. Ekelöf, M., Muddiman, D.C.: IR-MALDESI method optimization based on time-resolved measurement of ion yields. Anal. Bioanal. Chem. (2018). https://doi.org/10.1007/s00216- 017-0585-2

58. Khodjaniyazova, S., Nazari, M., Garrard, K.P., Matos, M.P.V., Jackson, G.P., Muddiman, D.C.: Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer Using Isotope Ratio Mass Spectrometry. Anal. Chem. (2018). https://doi.org/10.1021/acs.analchem.7b03983

59. Meija, J., Coplen, T.B., Berglund, M., Brand, W.A., De Bièvre, P., Gröning, M., Holden, N.E., Irrgeher, J., Loss, R.D., Walczyk, T., Prohaska, T.: Isotopic compositions of the elements 2013 (IUPAC Technical Report), (2016)

60. Alexandrov, T., Ovchinnikova, K., Palmer, A., Kovalev, V., Tarasov, A., Stuart, L.,

Nigmetzianov, R., Fay, D., Contributors, K.M.: METASPACE: A community-populated knowledge base of spatial metabolomes in health and disease. bioRxiv. (2019). https://doi.org/10.1101/539478

61. Ni, Z., Milic, I., Fedorova, M.: Identification of carbonylated lipids from different phospholipid classes by shotgun and LC-MS lipidomics. Anal. Bioanal. Chem. (2015). https://doi.org/10.1007/s00216-015-8536-2

62. Ejsing, C.S., Sampaio, J.L., Surendranath, V., Duchoslav, E., Ekroos, K., Klemm, R.W., Simons, K., Shevchenko, A.: Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. (2009). https://doi.org/10.1073/pnas.0811700106

63. Schuhmann, K., Almeida, R., Baumert, M., Herzog, R., Bornstein, S.R., Shevchenko, A.: Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes. J. Mass Spectrom. (2012). https://doi.org/10.1002/jms.2031

64. Makarov, A., Denisov, E., Kholomeev, A., Balschun, W., Lange, O., Strupat, K., Horning, S.: Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. (2006). https://doi.org/10.1021/ac0518811

CHAPTER 4

Direct Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) and Flash-Frozen Tissues

using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization

(IR-MALDESI) Mass Spectrometry Imaging

4.1 Introduction

Mass spectrometry imaging (MSI) is becoming a more established analytical tool in clinical and industrial laboratories due to advances in sensitivity, specificity, number of detected knowns and unknowns, spatial resolution, reproducibility, acquisition speed, and absolute quantification in some MSI systems.1,2,3 All these features help to characterize biological samples with a high level of detail.4,5, Mass spectrometry (MS) has already found its way into real operation rooms with the invention of the iKnife6 - a surgical knife that comes in direct contact with a biological tissue (e.g., patient) and immediately gives information to whether the tissue has cancerous cells or not. Therefore, it is only a matter of time before MSI platforms are utilized in a similar manner.

Today, the most common practice for evaluation of disease-affected tissues is formalin fixation that preserves cellular morphology and aids in diagnosis and prognosis of a disease. 7,8

With this technique, pathologists can characterize the shape, structure, and composition of cells in a tissue confirming or denying the presence of a disease.5 Although, the microscopic examination of fixed tissues is considered an invaluable “gold standard”7 for assessment of a disease, the results can be subjective due to a combination of external factors such as human interpretation, variability in staining quality, nature of the sample, indistinguishable histological features that are treated as abnormal.5 Therefore, there is a need for a complementary technology that would allow for more objective analyses.5

Recently, scientists in the MSI community have started showing an interest towards formalin-fixed and paraffin-embedded (FFPE) tissues because hospitals have a vast repositories of stored FFPE tissues, and also, obtaining some freshly-excised biological samples (especially from humans) can be really difficult, primarily because not a lot of patients are willing to donate their biopsies to research laboratories. For example, MALDI has been shown to be very successful in detection of a range of proteins, peptides, glycans, metabolites, and lipids from

FFPE tissues.9,10,11,12,13,14 Even though MSI of FFPE tissues exhibit changes in chemical nature of the sample, we are still curious to know if FFPE blocks can be useful in any way for soft ionization imaging sources such as IR-MALDESI because one of the main applications of IR-

MALDESI MSI is understanding human health and disease.

IR-MALDESI is an ambient ionization source that has been extensively used for direct analyses of fresh biological tissues (see Chapter 3 for details about the source operation).15 Prior to IR-MALDESI imaging, the sample undergoes no chemical treatments other than flash- freezing where ice plays the role of an energy-absorbing matrix. In Chapter 3, we discussed a range of lipid categories, classes, and subclasses (lipid nomenclature and classification see in the Introduction in Chapter 3) that can be successfully extracted and ionized using IR-

MALDESI source. Work discussed in Chapter 3 was of a very high interest because we know that in IR-MALDESI analyses, lipids are usually the most abundant molecular classes detected from fresh and unmodified biological tissues.16,17 And knowing that some lipids have already been successfully detected from FFPE tissues using MALDI13,14,18,12,19, we got very interested in understanding what lipids IR-MALDESI can extract and ionize from a soft FFPE tissue.

In this work we examine lipid classes and ions that can be confidently (MS/MS) detected from FFPE and flash-frozen (FF) rat liver sections using IR-MALDESI source coupled with a

Q Exactive Plus mass spectrometer. Our goal is to investigate the impact of formalin fixation on lipids detected in positive and negative mode across m/z 150-2000 range.

4.2 Methods

4.2.1 Materials

Optimal Cutting Temperature (OCT) embedding medium (for cryosectioning), ACS certified 2-methylbutane, OptimaTM LC/MS grade methanol and water were purchased from Fisher

Scientific (Pittsburgh, PA, USA). Acetic acid and formic acid were purchased from Sigma-Aldrich

(St. Louis, MO, USA). Nitrogen gas was obtained from Arc3 Gases (Raleigh, NC, USA) and plain microscope slides were purchased from VWR (Radnor, PA, USA). Flash-frozen liver piece was sliced on a Leica CM 1950 cryomicrotome (Buffalo Grove, IL, USA) using a high-profile coated microtome blade from VWR and (Batavia, IL, USA). Chamber temperature during sectioning was maintained at -15 °C. FFPE rat liver piece was sliced at room temperature. The thickness of all rat liver slices (both FF and FFPE) was 10 µm.

4.2.2 Rat Liver

Fresh liver was harvested from a healthy adult rat. The biggest liver lobe was cut in half: the first half was flash-frozen in isopentane/dry ice bath and the second half was formalin-fixed and paraffin-embedded. Rat liver pieces were cut longitudinally in 10 µm-thick slices, and then mounted on a plain, clean glass slide.

4.2.3 Flash-freezing Protocol

Procedures listed below (Figure 4.1) must be done in a fume hood for safety reasons: dry ice is made up of carbon dioxide (CO2), therefore, as dry ice pellets turn into CO2 gas, it is crucial to keep the room/working area well-ventilated to prevent the replacement of oxygen with carbon

dioxide.

• Place a beaker in the bottom of a Styrofoam container and fill the space around the beaker

with dry ice pellets.

• Place some pellets in the beaker and slowly add isopentane (2-methyl butane) and wait (3-

5 minutes) to let isopentane/dry ice bath to thermally equilibrate. When the pellets stop

bubbling vigorously, let the weigh boat (with tissue in it) float in the isopentane/dry ice

bath until the tissue is frozen. Be careful, do not let isopentane spill inside the weigh boat.

• Keep the tissue in -80°C freezer until the time of analysis. Remember to not store dry ice

pellets in the freezer/fridge as the air-tight container (e.g., fridge) with CO2 pellets in it

might explode.

Figure 4.1. Flash-freezing protocol. 4.2.4 Preparation of FFPE Tissue

Formalin is an alternative name for an aqueous solution of formaldehyde. Formaldehyde solution is considered a hazardous, carcinogenic compound whose vapor is toxic. Therefore, when working with formaldehyde solutions, it is required to always work in well-ventilated areas and wear personal protective equipment.

FFPE tissues for this study were prepared by technicians from NCSU Veterinary School

(Histopathology Laboratory, NCSU Veterinary School, Raleigh, NC).

• During preparation of FFPE tissue, the Tissue-Tek VIP 6 Processor was used to thoroughly

penetrate tissue sample with reagents (under vacuum and pressure). The sample

preparation consists of 4 main steps: fixation, dehydration, clearing, and infiltration.

During fixation process, tissue is soaked in 10% neutral buffered formalin (NBF). To

remove water during dehydration, sample is submerged in 70% ethanol. Clearing process

involves xylene (must be miscible with both alcohol and paraffin) that removes the residual

alcohol from the tissue. And finally, infiltration is permeation and saturation of tissue by

paraffin leading to matrix formation.

• Trim tissue so that it is no more than 4 mm thick.

• Transfer tissue to cassette labeled with a marker containing ink that is alcohol and xylene

resistant.

• Fix tissue for 24 hours in 10% NBF.

• Transfer tissue to 70% ethanol.

• Proceed with the rest of the processing steps or hold indefinitely in 70% ethanol.

4.2.5 IR-MALDESI MSI

The same IR-MALDESI parameters used in Chapter 3 were implemented in this study

(Figure 4.2).

Samples were analyzed in a DDA fashion (see Chapter 3 for details about the DDA method) across a wide m/z range using an IR-MALDESI source coupled with a Q Exactive Plus.

One hundred most abundant background ions (Table C.1) were included in the DDA exclusion list. Two mid-IR laser pulses with an incident energy of approximately 1 mJ per pulse were used for ablation events. Solvent for positive and negative ESI was 0.2% formic acid and 1 mM acetic acid in 50:50 (volume:volume) methanol:water, respectively. All imaging experiments were

72 carried out using an IR-MALDESI protocol for analyses of soft, flash-frozen tissues. Lipid ions and sub-classes were annotated using LipidSearchTM 4.2.21 software.

Figure 4.2. Experimental design.

4.3 Results and Discussion

The objective of this work is to investigate lipids that can be confidently extracted and ionized from a 10-µm-thick FFPE rat liver slice using IR-MALDESI. Fresh-frozen 10-µm-thick rat liver slice was used as a control. Both tissues (fresh and FFPE) were analyzed using the same

IR-MALDESI protocol and the imaging data was collected in a data-dependent acquisition (DDA) mode, where the most abundant tissue-specific ions were undergoing MS/MS fragmentation.

In this work we focus on lipid categories, classes, and subclasses. Nomenclature and classification of lipid species was discussed in detail in the Introduction of Chapter 3 of this thesis.

20,21,22,23 Briefly, there are eight (8) lipid categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, saccharolipids, polyketides; and each category has its own classes and subclasses.20 Lipids were annotated using LipidSearchTM 4.2.21 software from Thermo Scientific. LipidSearch reports quality metrics such as m-scores and grades. m-score is calculated based on the number of matches with the product ion: the higher the m-score the more accurate is identification. Grade indicates accuracy of identification: A is the highest and

D is the lowest grade. The list with all lipid classes that can be confidently (MS/MS) annotated by

LipidSearch are shown in the Table B.2.

We are targeting only lipids in this study because previous work shows that majority of lipid species are ionized successfully in the IR-MALDESI source (see Chapter 3 of this thesis for additional reference) and also because lipids are one of the most abundant molecular classes detected from biological samples when analyzed with IR-MALDESI source.24,16,25 Additionally, specific lipids can be very powerful diagnostic tools that help to distinguish heathy tissues from disease-affected tissue.26,27,28,29

FFPE and flash-frozen (FF) tissues show similar MS1 profiles as shown in Figure 4.3, where MS1 data was collected across two m/z ranges (150-600, 500-2000) and two polarities

(positive and negative). The reason behind collecting IR-MALDESI data in two difference m/z windows is to avoid a space-charge effect in the Orbitrap mass analyzer which might be caused by ions with very different kinetic energies (e.g., ion with m/z 200 vs. 1900).30,31,32 Spectral similarities can be clearly observed in negative mode, low mass range where there are no evident abundant peaks between m/z 350 and 600 in both FFPE and FF spectra. Similarly, high mass range, positive mode FFPE and FF spectra do not show any abundant peaks above m/z 1000. Labeled peaks in Figure 4.3 are lipid ions that were confidently identified with MS/MS meaning that every precursor ion has its corresponding MS/MS spectrum (Figure C.1) in the collected imaging data.

For full names of abbreviated lipids in Figure 4.3, please refer to Table B.2.

Before analyzing MS/MS data in more detail, we wanted to investigate common precursor ions that were present both in FFPE and FF data, because there is a possibility that some of the low-abundant lipid ions, from FFPE tissue, were not selected for MS/MS (in DDA) and therefore were labeled as “missed” ions that were actually present in FFPE but in very low concentrations.

Figure 4.3. Representative MS1 spectra collected from formalin-fixed paraffin-embedded (FFPE) and flash-frozen (FF) rat liver tissues.

Our data shows that FFPE and FF tissues have 823 common tissue-specific precursor ions that were present in MS1 data (Figure 4.4). Ion abundances were converted to the number of ions

(y-axis in Figure 4.4) by multiplying reported abundance (found in .RAW files) by the injection time (IT):33

number of ions = .RAW abundance (ions/sec) × IT (sec).

Figure 4.4. Abundance of 823 tissue-specific precursor ions that were detected both in FF and FFPE tissues. Blue circles represent lipid ions whose abundance was above recommended threshold of ~120 ions.

MSiReader34,35 imaging software was use for exclusions low-abundant peaks (< 120 ions) and background ions (Table C.1), as well as for rapid identification of the common 823 tissue- specific ions with m/z tolerance of ±2.5 ppm (part per million) for the peak of interest. Note that all precursor peaks with the abundances below recommended threshold of 120 ions were filtered out because peaks below this threshold might be produced by the thermal noise from the image- current preamplifier (ion-detection method in the Orbitrap mass analyzer).36 After fitting a line in the scattered plot of Figure 4.4, we came to a conclusion that abundance of all common ions detected from both FF and FFPE tissue is comparable due to a slope of 0.55: abundance of FFPE ions is 0.55 times less abundant than FF ions.

After carefully investigating MS1 data collected from FFPE and FF tissues, we moved onto

MS/MS data to work with confidently identified lipid ions. Table C.2 shows all lipid ions (along with grades and m-scores) that were confidently annotated using LipidSearchTM based on MS and

MS/MS data. Overall, complementary lipid ions were detected from FFPE and FF tissues (see

Figure 4.3 for visual reference). However, there are also lipids that were detected only in FFPE or only in FF. For example, in low mass range positive mode, cholesterol ester was detected only in fresh tissue; in high mass range positive mode, glycosphingolipid, phosphatidyl-serine, phosphatidyl-etahnol-amine,phosphotidyl-choline, neutral glycerolipid, and bis-methyl phosphatidyl-ethanolamine were detected only from fresh tissue; and finally, in high mass range negative mode, phosphatidic acid and cardiolipin were detected only from FFPE tissue while phosphatidyl-glycerol was detected only from fresh tissue. See Table B.2 for lipid abbreviations and full names.

Venn diagrams in Figure 4.5 summarizes confident annotations (based on MS/MS) in terms of lipid ions, IDs, and classes and shows that complementary lipid ions, IDs, and classes

Figure 4.5. Venn diagram showing the number of (left) lipid ions, (middle) lipid IDs, and (right) lipid classes that were annotated by LipidSearchTM 4.2.21. were detected from FFPE and FF samples. Briefly, thirty-seven lipid ions were detected only from fresh tissue, twelve ions were detected only from FFPE tissue, and five common lipid ions were detected from both tissues. Table C.2 and C.3 list all lipid ions and classes/IDs, respectively, detected from FFPE and flash-frozen rat liver tissues.

Figure 4.6. (Left) the number of lipid ions that were confidently annotated (MS/MS) by LipidSearchTM 4.2.21. (Right) the number of lipid ions that that were annotated both confidently (MS/MS) and putatively (MS1).

After confidently annotating lipid ions with MS/MS using LipidSearch and generating detailed Venn diagrams shown in Figure 4.5, the last question we wanted to answer is how many low-abundant lipids from FFPE tissue were not selected for DDA fragmentation? To answer this

78 question, all ions that were confidently annotated in FFPE tissue were searched against MS1 data of FF tissue and vice versa (Figure 4.6). This way we were looking for all confidently identified molecular ions that were detected from the same rat liver. Figure 4.6 (right) shows that 5 lipid ions confidently detected from FFPE can be recovered from fresh tissue; and similarly, 34 lipid

Figure 4.7. Abundances of 44 common lipid ions that were identified using MS2 data from either one or both FF and FFPE tissues. Orange circles represent lipid ions whose abundance was below recommended threshold (120 ions).

79 ions that were confidently annotated from fresh tissue can be recovered from FFPE tissue. Figure

4.6 (right) summarizes that 44 lipid ions were detected in both FFPE and FF rat liver slices.

To find out how abundance of 44 common lipid ions (detected either by MS1 or/and MS2) changed across tissues, we generated a plot were abundance of lipids detected in FF tissue was plotted against FFPE (Figure 4.7). Here ion abundance was converted to the number of ions, and ions with abundance of 120 ions or less were highlighted in orange circles. Linear function was fit in the scatter plot using only peaks whose abundance was above recommended threshold of 120 ions. Error bars represent 95% confidence intervals. Slope of the fitted line is equal to approximately 0.2 which indicates that lipid ions detected from FFPE tissues were about 5-fold less abundant than the same lipids detected from fresh tissue

4.4 Conclusions

In this work, we examined lipid ions, IDs, and classes that could be successfully extracted and ionized from a flash-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) rat liver slices using IR-MALDESI source coupled with Q Exactive PlusTM mass spectrometer. Analysis of FFPE tissue by IR-MALDESI was demonstrated feasible and abundance of all common ions detected from both FF and FFPE tissue is comparable. Lipids that were confidently annotated from FFPE tissue were about 5-fold less abundant in comparison to the same lipids detected from FF tissue.

Complementary lipid ions, IDs, and classes were detected from FFPE and flash-frozen tissues.

4.5 Acknowledgements

The authors acknowledge the financial support received from NIH (R01GM087964) and

North Carolina State University (NCSU). Measurements were made in the Molecular Education,

Technology, and Research Innovation Center (METRIC) at NCSU. The authors also thank NCSU

Histopathology Laboratory at Veterinary School for preparation of FFPE samples.

4.6 References

1. Vaysse, P. M., Heeren, R. M. A., Porta, T. & Balluff, B. Mass spectrometry imaging for clinical research-latest developments, applications, and current limitations. Analyst (2017) doi:10.1039/c7an00565b.

2. Addie, R. D., Balluff, B., Bovée, J. V. M. G., Morreau, H. & McDonnell, L. A. Current State and Future Challenges of Mass Spectrometry Imaging for Clinical Research. Anal. Chem. (2015) doi:10.1021/acs.analchem.5b00416.

3. Li, N. et al. Recent advances of ambient ionization mass spectrometry imaging in clinical research. Journal of Separation Science (2020) doi:10.1002/jssc.202000273.

4. Buchberger, A. R., DeLaney, K., Johnson, J. & Li, L. Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Analytical Chemistry (2018) doi:10.1021/acs.analchem.7b04733.

5. Leung, F. et al. Mass spectrometry-based tissue imaging: The next frontier in clinical diagnostics? Clinical Chemistry (2019) doi:10.1373/clinchem.2018.289694.

6. Alexander, J. et al. A novel methodology for in vivo endoscopic phenotyping of colorectal cancer based on real-time analysis of the mucosal lipidome: a prospective observational study of the iKnife. Surg. Endosc. (2017) doi:10.1007/s00464-016-5121-5.

7. Lemaire, R. et al. Direct analysis and MALDI imaging of formalin-fixed, paraffin- embedded tissue sections. J. Proteome Res. (2007) doi:10.1021/pr060549i.

8. Bauer, D. R., Otter, M. & Chafin, D. R. A New Paradigm for Tissue Diagnostics: Tools and Techniques to Standardize Tissue Collection, Transport, and Fixation. Current Pathobiology Reports (2018) doi:10.1007/s40139-018-0170-1.

9. Casadonte, R. & Caprioli, R. M. Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry. Nat. Protoc. (2011) doi:10.1038/nprot.2011.388.

10. Powers, T. W. et al. Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked Glycan expression in tissues. Anal. Chem. (2013) doi:10.1021/ac402108x.

11. Buck, A. et al. High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed, paraffin-embedded clinical tissue samples. J. Pathol. (2015) doi:10.1002/path.4560.

12. Ly, A. et al. High-mass-resolution MALDI mass spectrometry imaging of metabolites from formalin-fixed paraffin-embedded tissue. Nat. Protoc. (2016) doi:10.1038/nprot.2016.081.

13. Carter, C. L., McLeod, C. W. & Bunch, J. Imaging of phospholipids in formalin fixed rat

brain sections by matrix assisted laser desorption/ionization mass spectrometry. J. Am. Soc. Mass Spectrom. (2011) doi:10.1007/s13361-011-0227-4.

14. Pietrowska, M., Gawin, M., Polańska, J. & Widłak, P. Tissue fixed with formalin and processed without paraffin embedding is suitable for imaging of both peptides and lipids by MALDI-IMS. Proteomics (2016) doi:10.1002/pmic.201500424.

15. Khodjaniyazova, S., Hanne, N. J., Cole, J. H. & Muddiman, D. C. Mass spectrometry imaging (MSI) of fresh bones using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Anal. Methods (2019) doi:10.1039/c9ay01886g.

16. Xi, Y., Tu, A. & Muddiman, D. C. Lipidomic profiling of single mammalian cells by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Anal. Bioanal. Chem. (2020) doi:10.1007/s00216-020-02961-6.

17. Bagley, M. C., Ekelöf, M. & Muddiman, D. C. Determination of Optimal Electrospray Parameters for Lipidomics in Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging. J. Am. Soc. Mass Spectrom. (2020) doi:10.1021/jasms.9b00063.

18. Denti, V. et al. Antigen Retrieval and Its Effect on the MALDI-MSI of Lipids in Formalin-Fixed Paraffin-Embedded Tissue. J. Am. Soc. Mass Spectrom. (2020) doi:10.1021/jasms.0c00208.

19. Vos, D. R. N., Bowman, A. P., Heeren, R. M. A., Balluff, B. & Ellis, S. R. Class-specific depletion of lipid ion signals in tissues upon formalin fixation. Int. J. Mass Spectrom. (2019) doi:10.1016/j.ijms.2019.116212.

20. Fahy, E. et al. A comprehensive classification system for lipids. J. Lipid Res. (2005) doi:10.1194/jlr.E400004-JLR200.

21. Fahy, E., Cotter, D., Sud, M. & Subramaniam, S. Lipid classification, structures and tools. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids (2011) doi:10.1016/j.bbalip.2011.06.009.

22. Liebisch, G. et al. Shorthand notation for lipid structures derived from mass spectrometry. J. Lipid Res. (2013) doi:10.1194/jlr.M033506.

23. Fahy, E. et al. Update of the LIPID MAPS comprehensive classification system for lipids. Journal of Lipid Research (2009) doi:10.1194/jlr.R800095-JLR200.

24. Loziuk, P., Meier, F., Johnson, C., Ghashghaei, H. T. & Muddiman, D. C. TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR- MALDESI imaging of lipids and LC-MS/MS label-free proteomics. Anal. Bioanal. Chem. (2016) doi:10.1007/s00216-016-9421-3.

25. Nazari, M. & Muddiman, D. C. Polarity switching mass spectrometry imaging of healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Analyst (2016) doi:10.1039/c5an01513h.

26. Eberlin, L. S. et al. Cholesterol sulfate imaging in human prostate cancer tissue by desorption electrospray ionization mass spectrometry. Anal. Chem. (2010) doi:10.1021/ac9029482.

27. Miyamoto, S. et al. Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator. EBioMedicine (2016) doi:10.1016/j.ebiom.2016.03.033.

28. Lu, Q., Hu, Y., Chen, J. & Jin, S. Laser Desorption Postionization Mass Spectrometry Imaging of Folic Acid Molecules in Tumor Tissue. Anal. Chem. (2017) doi:10.1021/acs.analchem.7b00140.

29. Nizioł, J. et al. Surface-Transfer Mass Spectrometry Imaging of Renal Tissue on Gold Nanoparticle Enhanced Target. Anal. Chem. (2016) doi:10.1021/acs.analchem.6b01859.

30. Gorshkov, M. V., Good, D. M., Lyutvinskiy, Y., Yang, H. & Zubarev, R. A. Calibration function for the orbitrap FTMS accounting for the space charge effect. J. Am. Soc. Mass Spectrom. (2010) doi:10.1016/j.jasms.2010.06.021.

31. Kharchenko, A., Vladimirov, G., Heeren, R. M. A. & Nikolaev, E. N. Performance of orbitrap mass analyzer at various space charge and non-ideal field conditions: Simulation approach. J. Am. Soc. Mass Spectrom. (2012) doi:10.1007/s13361-011-0325-3.

32. Hu, Q. et al. The Orbitrap: A new mass spectrometer. Journal of Mass Spectrometry (2005) doi:10.1002/jms.856.

33. Khodjaniyazova, S. et al. Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer Using Isotope Ratio Mass Spectrometry. Anal. Chem. (2018) doi:10.1021/acs.analchem.7b03983.

34. Bokhart, M. T., Nazari, M., Garrard, K. P. & Muddiman, D. C. MSiReader v1.0: Evolving Open-Source Mass Spectrometry Imaging Software for Targeted and Untargeted Analyses. J. Am. Soc. Mass Spectrom. (2018) doi:10.1007/s13361-017-1809-6.

35. Robichaud, G., Garrard, K. P., Barry, J. A. & Muddiman, D. C. MSiReader: An open- source interface to view and analyze high resolving power MS imaging files on matlab platform. J. Am. Soc. Mass Spectrom. (2013) doi:10.1007/s13361-013-0607-z.

36. Makarov, A. et al. Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. (2006) doi:10.1021/ac0518811.

CHAPTER 5

Three-Dimensional Imaging with Infrared Matrix-Assisted

Laser Desorption Electrospray Ionization Mass Spectrometry

Reprinted with permission from: Bai, H.#, Khodjaniyazova, S.#, Garrard, K. P., and Muddiman, D. C. Journal of the American Society for Mass Spectrometry, 2019, 31(2), 292-297. #Authors contributed equally to this work. Copyright © 2019, American Chemical Society.

5.1 Introduction

Mass spectrometry imaging (MSI) reveals a glimpse into complex biological processes by simultaneously localizing different chemical species. MSI has been successfully applied in proteomics,1 lipidomics,2 metabolomics,3 pharmaceutical forensics,4 and other fields.5 For example, monitoring how a drug or disease marker is spatially distributed helps to understand the mechanism of a disease.6 Because biology is a three-dimensional phenomenon, three-dimensional mass spectrometry imaging (3D MSI) has the potential to uncover unique information that cannot be collected using traditional two-dimensional (2D) MSI approaches.

3D MSI has developed rapidly with emerging ionization methods such as matrix-assisted laser desorption ionization (MALDI),7 secondary ion mass spectrometry (SIMS),8 desorption electrospray ionization (DESI),9 and laser desorption ionization (LDI).10 There are two primary modes of 3D MSI: (1) serial-section-based and (2) ablation-based, with the former being more popular.11 In a typical 3D MSI study, the sample is sliced serially, and individual 2D MSI experiments are performed on each section. Then, consecutive 2D images are registered to reconstruct a 3D ion heat map for each analyte. This technique is an “add-on” to all regular mass spectrometry imaging methods because it puts no demand on instrumentation, whereas the real difficulty resides on sample preparation, registration of images, and data processing. Although many results have been reported using this protocol, a significant amount of sample is not analyzed

84 because imaging occurs in a series of discrete layers. Also, registration of 2D images to a common coordinate system can be challenging due to sample deformation during sectioning, orientation of the sample layers, and natural variation in tissue shape with depth. Because 3D images have to be reconstructed, computational algorithms have been developed to stack images based on the original morphological information obtained with magnetic resonance imaging (MRI) and histological staining.12,13 These data processing steps make 3D MSI a time-consuming method.

The problems mentioned above for serial-section-based 3D imaging can be avoided with ablation-based 3D MSI where no sectioning is needed. In the latter mode, an energetic ion or light beam ablates materials from the sample, continuously exposing a new surface for imaging in the x– y-plane. As ablation goes deeper, chemical changes across the z-direction are revealed in recorded mass spectra. Ablation-based MSI mode has been successfully applied in SIMS and laser ablation electrospray ionization (LAESI).14,15 3D SIMS MSI utilizes two beams: the first beam ejects atoms, molecules, and secondary ions from the surface while the second beam sputters the already analyzed surface to create a new plane for imaging the next layer.16 These two ion beams are used iteratively to create 3D chemical maps of species. Based on this protocol, Castellanos and coworkers revealed that triacylglycerides were abundant in oocyte region of sugar-fed female Aedes aegypti mosquitoes.17 Despite high spatial resolution in both the z- and x–y- directions,18 SIMS is not as soft as other ionization sources such as MALDI and ESI. Moreover,

SIMS requires a vacuum condition, so it cannot be used to investigate volatile molecules.

Alternatively, Nemes et al. used a laser to ablate material in a 3D manner with an ambient ionization technique named LAESI.15 LAESI 3D MSI was achieved by creating depth profiles at each grid point in a region of interest (ROI). Six-layer 3D images for metabolites in Iynise leaf tissue were constructed with resolution of approximately 300 and 30 μm in the x–y- and z-

85 directions, respectively.

For the first time, we used infrared matrix-assisted laser desorption electrospray ionization

(IR-MALDESI) for 3D MSI. IR-MALDESI was introduced by Muddiman in 200619 and is an atmospheric ionization source that combines features of laser desorption and electrospray ionization (ESI). Neutral materials are desorbed by an infrared laser and are then partitioned into charged droplets generated by an orthogonal ESI. IR-MALDESI allows for direct detection with a lateral resolution of 50 μm without oversampling.20 IR-MALDESI has been used for 2D imaging of neurotransmitters,21 peptides,22 and other species,23 whereas 3D IR-MALDESI MSI has never been demonstrated before.

In this work, 3D IR-MALDESI MSI was performed by repeatedly collecting 2D images over the same ROI. In this proof-of-principle experiment, over-the-counter (OTC) pharmaceuticals produced in the form of multiple unit pellet system (MUPS) were used as models to test the feasibility of IR-MALDESI for 3D MSI. Spherical components inside each pill (Figure 5.1C) have different chemical compositions compared to that of surrounding powder and can be spatially resolved in 3D MSI. Depth resolution was explored on a full pill followed by 2D and 3D MSI performed on a half pill.

5.2 Experimental Section

5.2.1 Materials

Prilosec OTC (Cincinnati, OH, United States), an omeprazole delayed-release tablet, was purchased from a local pharmacy. All of the tablets used in this study were produced in the lot number 8124171971. In the first part of the experiment, which determines depth resolution on a full pill (Figure 5.1A), no sample preparation was needed prior to imaging. In the second part of the experiment, the tablet was trimmed flat (Figure 5.1B) with a Leica CM1950 cryomicrotome

(Buffalo Grove, IL, United States) for 2D and 3D MSI. HPLC-grade methanol and water were purchased from Burdick and Jackson (Muskegon, MI, United States). MS-grade formic acid was purchased from Sigma-Aldrich (St. Louis, MO, United States). Burn paper for laser focusing was purchased from ZAP-IT (ZFC-23; ZAP-IT, Concord, NH, United States).

5.2.2 IR-MALDESI System

Figure 5.1. Pills used as a model for demonstration. (A) Optical image of a full pill, where the z- resolution at different energy levels was determined. (B) Optical image of a pill trimmed in half for 2D and 3D MSI; small circles are due to MUPS formulation. (C) Schematic of a pellet and its components, where each color indicates different components.

An IR-MALDESI source coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher

Scientific, Bremen, Germany) was used for all the imaging experiments presented. The set-up has been previously described in detail by others.24 In short, neutrals ablated by laser partition into charged droplets from an orthogonal electrospray are sampled into the mass spectrometer with an injection time of 25 ms and automatic gain control (AGC) disabled. Mass spectra were collected across m/z 150–600 in positive mode with lock mass at m/z 371.1012 and mass resolving power

140 000 at m/z 200. Electrospray flow-rate was set to be 2 μL/min. A focused laser (IR-Opolette

2731, Opotek, Carlsbad, CA, United States) with a wavelength of 2940 nm was fired one laser pulse at each point within the region of interest (ROI). Repetition rate was set to be 20 Hz. More

87 information about the IR-MALDESI interface can be found in Table D.1

5.2.3 Depth Resolution Determination

Laser energies at 0.3 and 1.2 mJ/pulse were used to investigate dependence of ablation depths on laser energy. Laser energy was adjusted using a Q-switch which triggers laser pulses

(Table D.2). Energy at target was measured with a laser power meter (Nova 2, Ophir, Jerusalem,

Israel). Depth profiles after ablation of more than one layer were accurately measured with a confocal laser scanning microscope (VK-X1100, Keyence, Itasca, IL, United States). Depth profiles were used to calculate depth resolution, or in other words, depth of ablation region after firing one laser pulse per voxel.

5.2.4 2D MSI

The trimmed flat tablet was adhered to the imaging stage using a double-sided tape. ROI on the half pill was 20 × 45 voxels with a spot-to-spot spacing of 150 μm. Laser energy at target was 1.2 mJ/pulse. We chose the highest energy level for 2D MSI to mimic parameters that we usually use for 2D IR-MALDESI MSI.

5.2.5 3D MSI

3D MSI was performed on a trimmed pill at a ROI of 15 × 15 voxels with a step size of 52

μm, using one of the lowest laser energies (0.3 mJ/pulse) to generate highest possible spatial resolution in z-direction. Prior to 3D IR-MALDESI, we estimated depth resolution by measuring depth profiles from ablation of 5 and 10 layers without stage height adjustment. To preserve laser focus during ablation of 50 layers in 3D IR-MALDESI MSI, the stage was moved up manually after analysis of every 3 layers.

5.2.6 Data Processing

XCalibur raw data were converted to imzML format using an open source application raw

88 to imzML converter.25 Then, imzML files were analyzed with MSiReader, a Matlab-based software developed in the Muddiman group.26,27

5.3 Results and Discussion

5.3.1 Depth Resolution Determination on a Pill

Spatial resolution dictates the quality of generated ion heat maps. Depth resolution was determined by measuring the depth of ablated spots. Lower laser energy and denser analyzed surface result in higher depth resolution (less material ablated per laser shot). Depth resolutions at the highest (1.2 mJ) and lowest (0.3 mJ) energy levels were explored. Optical and laser images after ablation of 1, 5, 10, 15, and 20 layers were measured with confocal laser scanning microscopy

(Figures 5.2A and 5.2C) and show that the ablation depth increased with the number of ablated layers. From side views of laser images in Figures 5.2A and 5.2C, the crater was deeper in the center compared to the edges due to the Gaussian nature of the beam. The ablation depth was measured by averaging depth profiles from 20 lines (at 2 μm increments) drawn across each ROI.

Depth profiles show a linear relationship with layer numbers for both energy levels (Figures 5.2B and 5.2D). The average thickness of the outer pink enteric coating (65 μm) was measured using a

Leica optical microscope. Three and 30 layers had to be ablated to completely desorb the outer layer with the laser energy of 1.2 and 0.3 mJ/pulse, respectively. This is consistent with optical results shown in Figures 5.2A and 5.2C, where white powder shows up after ablation of 5 layers at the 1.2 mJ/pulse energy level but never appears within 20 layers ablated at the 0.3 mJ/pulse energy level.

5.3.2 2D MSI on a Pill Microtomed in Half

2D MSI was performed on the half pill to resolve the chemical compositions of the pill.

Three pill-specific molecules (Figure 5.3) were found inside the pill and used for demonstration

of 3D IR-MALDESI MSI. Monomers of starch (C6H10O5, m/z 163.0601) were distributed in the core of pellet as well as the surrounding powder outside pellets (Figure 5.4A); triethyl citrate

(C12H20O7, m/z 277.1282), a marker of enteric coating for delaying drug release, was distributed in the outer layer of pellet and surrounding powder among the pellets (Figure 5.4B), and omeprazole (C17H19N3O3S, m/z 346.1220), the active ingredient, was distributed in the inner layer of the pellet (Figure 5.4C). These three markers were successfully resolved in a half pill (Figure

5.4I), and their colocalization (Figure 5.4D) agrees with the pellet’s model scheme shown in

Figure 5.1C.

Figure 5.2. Ablation depth, optical, and laser images were obtained using a confocal laser scanning microscope after ablation of 1, 5, 10, 15, and 20 layers. Optical and laser images show profiles after ablation at (A) 1.2 and (C) 0.3 mJ/pulse. Color scale bars show ablation depth in micrometers. The linear relationship between ablation depth and layer number is shown for two laser energy levels: (B) 1.2 and (D) 0.3 mJ/pulse. Depth resolution (depth of ablated region per layer) was estimated as weighted ablated depth: 20.1 μm for 1.2 mJ/pulse and 2.3 μm for 0.3 mJ/pulse. Spot size was 100 μm at 1.2 mJ/pulse and 78 μm at 0.3 mJ/pulse.

Figure 5.3. Representative mass spectra for (A) active ingredient omeprazole (C17H19N3O3S), (B) triethyl citrate (C12H20O7), and (C) monomers of starch (C6H10O5). MMA denotes mass measurement accuracy.

Figure 5.4. Three representative molecules were selected to show their distributions in the half pill: (A) starch (m/z 163.0601), (B) triethyl citrate (m/z 277.1282), and (C) active ingredient omeprazole (m/z 346.1220). Panel D shows a colocalization of three molecules. Panels E and F 13 34 show the distribution of A + 1 C1 (m/z 347.1253) and A + 2 S1 (m/z 348.1178) isotopologues of omeprazole, respectively. Panel G shows a colocalization of monoisotopic peak A (red), A + 13 34 1 C1 (green), and A + 2 S1 (blue) isotopologues of omeprazole with the same gain. (I) Optical image of ROI. The laser spot size was 120 μm.

34 13 For the A + 2 peaks of omeprazole, S1 (m/z 348.1176) and C2 (m/z 348.1291) isotopologues were baseline resolved (Figure 5.3A). We can count the number of sulfurs, and this approach allows for the determination of unique elemental compositions.28,29 It is important to note

92 that, although sulfur counting was not required here, given that the components in the OTC are known, it is a very useful for unknowns. The number of sulfurs in omeprazole was calculated using

Equation 5.1 and was estimated to be 1.0,28−30 using a theoretical sulfur abundance of 0.042 as reported by IUPAC:31

퐴+2 푠푢푙푓푢푟 푛표. = 푒푥푝푒푟𝑖푚푒푛푡푎푙 /0.042 Equation 5.1 퐴푒푥푝푒푟𝑖푚푒푛푡푎푙

13 Also, the distributions of A + 1 C1 (m/z 347.1253, Figure 5.4E) and A +

34 2 S1 (m/z 348.1178, Figure 5.4F) isotopologues of omeprazole colocalize with the monoisotopic peak (Figure 5.4G) with the same gain, confirming the sensitivity of the imaging method.

5.3.3 3D MSI on the Pill Microtomed in Half

3D MSI was performed on a trimmed pill using the lowest stable energy level of 0.3 mJ/pulse (Table D.2). Prior to the 3D experiment, 5 and 10 layers (Figures D.1 and D.2, respectively) were ablated from the hall pill without stage height adjustments to determine the depth resolution, which was calculated as the weighted average of 16.3 μm with ablation depth of

83.1 μm for 5 layers and 161.0 μm for 10 layers. To preserve laser focus during ablation of 50 layers in 3D fashion, the height of the translational stage was adjusted after every 3 ablated layers.

We did not adjust the stage height after each ablated layer because of the stage precision in the z- direction (Table D.2). One tick mark on the adjustment knob is 25 μm, while depth resolution is

16.3 μm per layer. After every 3 layers imaged (16.3 × 3 = 48.9 μm), we moved the stage up by 2 tick marks (∼50 μm). This is the smallest increment we could achieve for this experiment. Ablation depth after 50 layers was measured as 840.4 μm with an average depth resolution of 16.3 μm/layer with stage height adjustment, compared to 7.9 μm/layer without stage height adjustment.

The heat maps for three representative molecules (Figure 5.5) and their colocalization

(Figure D.3B) show the process that one pellet was trimmed away and then another pellet was

93 imaged. The distribution of 34S isotopologue (Figure D.3A) confirms the presence of omeprazole across 50 layers. Constructed 3D heat maps (Figure 5.6) show the distribution of three representative markers in 3D space.

Figure 5.5. Ion abundance and distribution for A peaks of (A) starch, (B) triethyl citrate, and (C) omeprazole on the half pill across 50 layers with laser spot size 80 μm and depth resolution 16.3 μm.

Figure 5.6. Three-dimensional intensity maps for (A) starch, (B) triethyl citrate, and (C) omeprazole. (D) Colocalization of three markers in the pill with laser spot size 80 μm and depth resolution 16.3 μm.

In this work, we demonstrated utility of IR-MALDESI for 3D MSI, which offers an

alternative method to traditional 3D MSI. No excessive sample preparation steps or assistive

morphological information (e.g., MRI) is needed for imaging registration. The main source of

variance comes from inconsistent laser ablation performance from layer to layer, resulting in sharp

ablation profiles, as shown in Figures 5.2A and 5.2C. This is due to the Gaussian laser beam,

where higher energy is distributed in the center. Inconsistent ablation profiles could be avoided

with a beam homogenizer, which creates uniform beam profiles. However, the objective of this

work was to test that the current IR-MALDESI system is applicable for 3D MSI, rather than to

develop a robust method for 3D IR-MALDESI.

All in all, the presented work provides workflow for ablation-based 3D IR-MALDESI by presenting 3D MSI analysis on a 15 × 15 ROI across 50 layers within 70 min. IR-MALDESI has proven to be feasible for 3D MSI on hard OTC tablets. Future work will be focused on development of 3D IR-MALDESI MSI for analyses of biological samples.

5.4 Conclusions

OTC tablet formulated with MUPS technologies was analyzed using IR-MALDESI source coupled to a Q Exactive Plus mass spectrometer to evaluate the feasibility of the former for ablation-based 3D MSI. We also investigated the depth of ablated spots at different laser energy levels to determine a range of depth resolution. The highest and lowest depth resolutions were measured as 2.3 μm at 0.3 mJ/pulse and 20.1 μm at 1.2 mJ/pulse, respectively. Minimal to no sample preparation makes IR-MALDESI an alternative MSI tool for analyses of biological samples. Therefore, future work will be focused on developing reproducible methods for 3D IR-

MALDESI MSI of biological tissues.

5.5 Acknowledgments

The authors gratefully acknowledge the financial support received from NIH

(R01GM087964) and North Carolina State University. All mass spectrometry measurements were made in the Molecular Education, Technology, and Research Innovation Center (METRIC) at

North Carolina State University. Measurements using the confocal laser scanning microscope were performed at the Analytical Instrumentation Facility (AIF) at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation (Award

ECCS-1542015).

5.6 References

1. Cordero Hernandez, Y.; Boskamp, T.; Casadonte, R.; HaubergLotte, L.; Oetjen, J.; Lachmund, D.; et al. Targeted Feature Extraction in MALDI Mass Spectrometry Imaging to Discriminate Proteomic Profiles of Breast and Ovarian Cancer. Proteomics: Clin. Appl. 2019, 13, 1700168.

2. Garikapati, V.; Karnati, S.; Bhandari, D.R.; Baumgart-Vogt, E.; Spengler, B. High- Resolution Atmospheric-Pressure MALDI Mass Spectrometry Imaging Workflow for Lipidomic Analysis of Late Fetal Mouse Lungs. Sci. Rep. 2019, 9, 3192.

3. Tian, X.; Zhang, G.; Zou, Z.; Yang, Z. Anticancer Drug Affects Metabolomic Profiles in Multicellular Spheroids: Studies Using Mass Spectrometry Imaging Combined with Machine Learning. Anal. Chem. 2019, 91, 5802−5809.

4. (4) Galhena, A. S.; Harris, G. A.; Nyadong, L.; Murray, K. K.; Fernandez, F. M. Small Molecule Ambient Mass Spectrometry Imaging by Infrared Laser Ablation Metastable- Induced Chemical

5. Ionization. Anal. Chem. 2010, 82, 2178−2181.

6. (5) Buchberger, A.R.; DeLaney, K.; Johnson, J.; Li, L. Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Anal. Chem. 2018, 90, 240−265.

7. (6) Hamerly, T.; Everett, J.A.; Paris, N.; Fisher, S.T.; Karunamurthy, A.; James, G.A.; et al. Detection of Pseudomonas Aeruginosa Biomarkers from Thermally Injured Mice In Situ Using Imaging Mass Spectrometry. Anal. Biochem. 2017, 539, 144−148.

8. (7) Jones, E.E.; Quiason, C.; Dale, S.; Shahidi-Latham, S.K. Feasibility Assessment of a MALDI FTICR Imaging Approach for the 3D Reconstruction of a Mouse Lung. J. Am. Soc. Mass Spectrom. 2017, 28, 1709−1715.

9. (8) Vanbellingen, Q.P.; Castellanos, A.; Rodriguez-Silva, M.; Paudel, I.; Chambers, J.W.; Fernandez-Lima, F.A. Analysis of Chemotherapeutic Drug Delivery at the Single Cell Level Using 3D-MSITOF-SIMS. J. Am. Soc. Mass Spectrom. 2016, 27, 2033−2040.

10. (9) Lanekoff, I.; Burnum-Johnson, K.; Thomas, M.; Cha, J.; Dey, S.K.; Yang, P.; et al. Three-Dimensional Imaging of Lipids and Metabolites in Tissues by Nanospray Desorption Eectrospray

11. Ionization Mass Spectrometry. Anal. Bioanal. Chem. 2015, 407, 2063−2071.

12. (10) Kuznetsov, I.; Filevich, J.; Dong, F.; Woolston, M.; Chao, W.; Anderson, E.H.; et al. Three-Dimensional Nanoscale Molecular Imaging by Extreme Ultraviolet Laser Ablation Mass Spectrometry. Nat. Commun. 2015, 6, 6944.

13. (11) Palmer, A.D.; Alexandrov, T. Serial 3D Imaging Mass Spectrometry at its Tipping Point. Anal. Chem. 2015, 87, 4055−4062.

14. (12) Thiele, H.; Heldmann, S.; Trede, D.; Strehlow, J.; Wirtz, S.; Dreher, W.; et al. 2D and 3D MALDI-Imaging: Conceptual Strategies for Visualization and Data Mining. Biochim. Biophys. Acta, Proteins Proteomics 2014, 1844, 117−137.

15. (13) Oetjen, J.; Aichler, M.; Trede, D.; Strehlow, J.; Berger, J.; Heldmann, S.; et al. MRI- Compatible Pipeline for Three-Dimensional MALDI Imaging Mass Spectrometry Using PAXgene Fixation. J. Proteomics 2013, 90, 52−60.

16. (14) Bich, C.; Havelund, R.; Moellers, R.; Touboul, D.; Kollmer, F.; Niehuis, E.; et al. Argon Cluster Ion Source Evaluation on Lipid Standards and Rat Brain Tissue Samples. Anal. Chem. 2013, 85, 7745−7752.

17. (15) Nemes, P.; Barton, A.A.; Vertes, A. Three-Dimensional Imaging of Metabolites in Tissues under Ambient Conditions by Laser Ablation Electrospray Ionization Mass Spectrometry. Anal. Chem. 2009, 81, 6668−6675.

18. (16) Passarelli, M.K.; Pirkl, A.; Moellers, R.; Grinfeld, D.; Kollmer, F.; Havelund, R. The 3D OrbiSIMS-Label-Free Metabolic Imaging with Subcellular Lateral Resolution and High Mass-Resolving Power. Nat. Methods 2017, 14, 1175.

19. (17) Castellanos, A.; Ramirez, C.E.; Michalkova, V.; Nouzova, M.; Noriega, F.G.; Fernandez-Lima, F. Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D- SIMS) of Aedes Aegypti Ovarian Follicles. J. Anal. At. Spectrom. 2019, 34, 874−883.

20. (18) Bruinen, A.L.; Fisher, G.L.; Balez, R.; van der Sar, A.M.; Ooi, L.; Heeren, R. M. A. Identification and High-Resolution Imaging of αTocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2018, 29, 1571−1581.

21. (19) Sampson, J.S.; Hawkridge, A.M.; Muddiman, D.C. Generation and Detection of Multiply-Charged Peptides and Proteins by MatrixAssisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2006, 17, 1712−1716.

22. (20) Bokhart, M.T.; Manni, J.; Garrard, K.P.; Ekelof, M.; Nazari, M.; Muddiman, D.C. IR- MALDESI Mass Spectrometry Imaging at 50 Micron Spatial Resolution. J. Am. Soc. Mass Spectrom. 2017, 28, 2099−2107.

23. (21) Bagley, M.C.; Ekelöf, M.; Rock, K.; Patisaul, H.; Muddiman, D.C. IR-MALDESI Mass Spectrometry Imaging of Underivatized Neurotransmitters in Brain Tissue of Rats Exposed to Tetrabromobisphenol A. Anal. Bioanal. Chem. 2018, 410, 7979−7986.

24. (22) Fideler, J.; Johanningsmeier, S.D.; Ekelof, M.; Muddiman, D.C. Discovery and Quantification of Bioactive Peptides in Fermented Cucumber by Direct Analysis IR- MALDESI Mass Spectrometry and LC-QQQ-MS. Food Chem. 2019, 271, 715−723.

25. (23) Nazari, M.; Malico, A.A.; Ekelöf, M.; Lund, S.; Williams, G.J.; Muddiman, D.C. Direct Analysis of Terpenes from Biological Buffer Systems Using SESI and IR- MALDESI. Anal. Bioanal. Chem. 2018, 410, 953−962.

26. (24) Bokhart, M.T.; Muddiman, D.C. Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging Analysis of Biospecimens. Analyst 2016, 141, 5236−5245.

27. (25) Schramm, T.; Hester, Z.; Klinkert, I.; Both, J.P.; Heeren, R. M. A.; Brunelle, A.; et al. ImzML- A Common Data Format for the Flexible Exchange and Processing of Mass Spectrometry Imaging

28. Data. J. Proteomics 2012, 75, 5106−5110.

29. (26) Robichaud, G.; Garrard, K.P.; Barry, J.A.; Muddiman, D.C. MSiReader: An Open- Source Interface to View and Analyze High Resolving Power MS Imaging Files on Matlab Platform. J. Am. Soc. Mass Spectrom. 2013, 24, 718−721.

30. (27) Bokhart, M.T.; Nazari, M.; Garrard, K.P.; Muddiman, D.C. MSiReader v1.0: Evolving Open-Source Mass Spectrometry Imaging Software for Targeted and Untargeted Analyses. J. Am. Soc. Mass Spectrom. 2018, 29, 8−16.

31. (28) Khodjaniyazova, S.; Nazari, M.; Garrard, K.P.; Matos, M. P. V.; Jackson, G.P.; Muddiman, D.C. Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer Using Isotope Ratio Mass Spectrometry. Anal. Chem. 2018, 90, 1897−1906.

32. (29) Nazari, M.; Muddiman, D.C. Polarity switching mass spectrometry imaging of healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray

33. ionization (IR-MALDESI). Analyst 2016, 141, 595−605.

34. (30) Shi, S. D. H.; Hendrickson, C.L.; Marshall, A.G. Counting Individual Sulfur Atoms in a Protein by Ultrahighresolution Fourier Ransform Ion Cyclotron Resonance Mass Spectrometry: Experimental Resolution of Isotopic Fine Structure in Proteins. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 11532−11537.

35. (31) Meija, J.; Coplen, T.B.; Berglund, M.; Brand, W.A.; De Bievre, P.; Gröning, M.; et al. Isotopic Compositions of the Elements 2013 (IUPAC Technical Report). Pure Appl. Chem. 2016, 88, 293−306.

CHAPTER 6

Mass Spectrometry Imaging (MSI) of Fresh Bones using Infrared Matrix-Assisted Laser

Desorption Electrospray Ionization (IR-MALDESI)

The following work was reprinted with permission from the Royal Society of Chemistry: Khodjaniyazova, S., Hanne, N. J., Cole, J. H., and Muddiman, D. C. Analytical Methods, 2019, 11, 5929-5938. (This work was highlighted on the journal cover). Copyright © 2019, Royal Society of Chemistry.

6.1 Introduction

To understand a disease in a complex biological system, one should know not only molecular features characteristic to that specific illness but also their spatial distribution within an organism or an organ.1,2 Mass spectrometry imaging (MSI) is an analytical tool used for localization of molecular features and their relative abundances in a given sample. In a typical two- dimensional (2D) mass spectrometry imaging (MSI) experiment, the sample is sectioned first followed by collection of one mass spectrum from each spot on the slice. Next, imaging software is used to generate heatmaps showing the spatial distribution of ions and their abundances across a region of interest (ROI).3–5 One of the main advantages of MS-based imaging over other methods of chemical visualization is the label-free detection of a wide range of endogenous known and unknown compounds that can be detected simultaneously, provided they fall within the mass-to- charge (m/z) window of the mass analyzer and form ions with a charge of the same sign. Although

MSI is generally considered a qualitative analytical technique due to a significant signal variability, there are MSI systems that can accurately quantify analytes by depositing standards with a known concentration on top or underneath thin tissue sections.6,7 A myriad of commercial and home-built MSI systems are available today; however, a comprehensive review of instrumentation is beyond the scope of this article.

A typical MSI system consists of an ionization source and a mass spectrometer (MS).

100

Ionization source ‘extracts’ and ionizes biomolecules from the sample's surface, and their m/z ratios are measured in a mass spectrometer. Ionization source dictates what molecular classes will be extracted from a sample, and the quality of mass spectra depends on a mass analyzer. No single ionization source can extract and ionize absolutely all species present in a sample. In fact, each ionization source has its own limitations: for example, IR-MALDESI is rarely used for protein imaging in biological samples because highly-abundant lipids suppress protein signal.8 Therefore, sometimes MSI is used in tandem with other analytical techniques, such as LC-

MS, to unravel a more detailed qualitative and quantitative information about a biological sample.7,9

Preparation of biological samples is one of the most crucial factors influencing the quality and authenticity of MSI results. Prior to 2D MSI, the sample is usually cut in thin slices to ensure uniform sampling. Soft tissues can be easily sectioned using a cryomicrotome; however, sectioning hard tissues like fresh, unmodified mouse bones is challenging because bone section tends to curl or fall apart.10 The term ‘unmodified bone’ refers to a fresh bone that has not been chemically modified (no fixation, decalcification, infiltration steps, etc.) and will be used throughout the manuscript.

MSI of bones is a developing field and to date only secondary-ion mass spectrometry

(SIMS) and matrix-assisted laser desorption ionization (MALDI) have been reported in the literature as ionization sources compatible with MSI of bones.11–14 However, for any laser- or ion beam-based MSI approach, the bone sample has to be both flat and demineralized. To satisfy these requirements, bone undergoes a series of chemical treatments that change its molecular composition.10–12,14–18 Standard sample preparation steps usually involve at least one of the following procedures: decalcification to make bone softer, embedding to minimize destruction of

101 bone sections, and grinding to a desired thickness to create a flat and smooth bone segment.17 Each of these steps alters bone chemistry: decalcification removes all bone minerals,17 traditional embedding approaches can distort bone architecture,17 and grinding generates heat and can smear the bone surface.19,20 Therefore, developing a method for direct analysis of unmodified bones is critical for identification of biochemical signatures unique to healthy and disease-affected bones.

This work reports a sample preparation protocol for direct analysis of unmodified mouse bones using IR-MALDESI MSI.8,21 IR-MALDESI is a soft ionization source that has been used in

MSI to monitor spatial distributions of different molecules in biological7,9,22–27 and non-biological samples.28,29 IR-MALDESI combines the properties of ESI and MALDI and has several distinctive characteristics: prior to analysis, the sample undergoes no chemical modifications;30 ice is used as the energy-absorbing matrix;21 ionization takes place at ambient temperature and pressure; and multiple- and singly-charged ions can be generated by the source.31 Among all ionization sources available today, IR-MALDESI is the only source that tolerates samples with high salt concentrations,23,32 and inorganic minerals comprise approximately 70% of bone by weight.33 The fundamentals and operation of the IR-MALDESI source have been described elsewhere.10 Briefly, when imaging soft tissues, the sample is sliced in 5- to 200 μm-thick sections using a cryomicrotome and thaw-mounted onto a glass slide, which is then placed onto a mobile and

Peltier-cooled IR-MALDESI stage. After a thin ice layer is formed on top of the sample, a mid-IR laser pulse (2940 nm) is fired one spot at a time to ablate primarily neutral species by excitation of O–H stretching modes.34,35 These neutrals are ejected orthogonal to the surface and partition into charged ESI droplets. A decreasing pressure gradient draws charged droplets into a mass spectrometer where they undergo desolvation and charge transfer in an ESI-like manner.34 Next, the translational stage moves by a fixed distance in the x or y direction for analysis of adjacent

102 spots. Here we aimed to investigate the feasibility of the IR-MALDESI source for MSI and direct analysis of mouse bones. Our strategy was built around IR-MALDESI's tolerance for salts. We propose a simple and robust sample preparation protocol that helps to preserve physiological and chemical properties of bone prior to imaging.

6.2 Experimental

6.2.1 Mouse Bones

All animal procedures were approved by the North Carolina State University Institutional

Animal Care and Use Committee. Two C57Bl/6J mice (the Jackson Lab, Bar Harbor, ME, USA) received either a stroke (n = 1, female) or sham (n = 1, male) surgery at 22 weeks of age. Ischemic stroke was induced using the middle cerebral artery occlusion method,36,37 in which a silicone- tipped filament was inserted to the lumen of the right middle cerebral artery for 30 minutes, producing a mild-to-moderate strength stroke.38 For the sham surgery, the neck incision was made and remained open for 30 minutes, but the filament was not inserted into the artery.38 Mice recovered from stroke or sham surgery for four weeks, then were sacrificed by CO2 asphyxiation followed by cervical dislocation. Left humeri (paretic limbs affected by stroke) were removed and immediately flash-frozen.

Note that reported work is not a biological study but a protocol for a direct analysis and

MSI of bone tissues. Therefore, analysis of a limited number of bones derived from animals of different sex is acceptable.

6.2.2 Flash-freezing

The following protocol was used throughout the study for flash-freezing of bones:

(1) Pour liquid nitrogen into a lab ice bucket to a height of 5 cm. Pour isopentane (Burdick and Jackson, Muskegon, MI, USA) into a metal beaker to a height of 5 cm and slowly lower the

103 metal beaker into the ice bucket. The isopentane is ready to flash-freeze bone when it thickens, just prior to appearing milky.

(2) Remove as much soft tissue from the bone sample as possible. Use sterile forceps to submerge the bone sample in isopentane for 15–30 seconds. The sample is done freezing when any remaining soft tissue loses color.

(3) Gently remove the bone sample from the forceps. Immediately transfer the bone to an individually labeled plastic sample tube and store at −80 °C until analysis.

6.2.3 Cryosectioning

Prior to cryosectioning, whole mouse humerus was embedded in Plaster of Paris (DAP

Products Inc., Baltimore, MD, USA) to avoid direct contact of bone with optimal cutting temperature (OCT) compound, which can contaminate the front end of a mass spectrometer.

Embedding was done inside a CM 1950 cryomicrotome chamber (Leica Biosystems, Buffalo

Grove, IL, USA) at −20 °C. To prepare embedding mixture for cryosectioning, 2 parts (i.e., by volume) of Plaster of Paris were mixed with 1 part of Optima™ LC/MS grade water (Fisher

Scientific, Pittsburgh, PA, USA). This mixture was then poured in a square, disposable peel-a-way block (Polysciences Inc., Warrington, PA, USA). Plaster of Paris should be handled in a well- ventilated area (i.e., fume hood) to prevent inhalation of the dust. Frozen mouse bone (whole humerus) was carefully embedded in the Plaster of Paris medium in a desired orientation using pre-cleaned forceps followed by freezing of the Plaster cube (peel-a-way mold, Plaster of Paris, and whole bone) at −20 °C in the cryomicrotome chamber or on dry ice. After the Plaster cube

(peel-a-way mold, Plaster of Paris, and whole bone) had frozen, a thin layer of OCT compound

(Leica Biosystems, Buffalo Grove, IL, USA) was applied to a 40 mm (diameter) specimen disk

(Leica Biosystems, Buffalo Grove, IL, USA), and the Plaster cube was gently placed on the OCT-

104 coated disk. The disk and Plaster cube equilibrated for 10 minutes on the Peltier-cooled sample holder inside the cryomicrotome. Roughly half of the bone was carefully trimmed on a CM 1950 cryomicrotome (Leica Biosystems, Buffalo Grove, IL, USA) at −20 °C using a Surgipath high- profile disposable blade (Leica Biosystems, Buffalo Grove, IL, USA). Trimming was done in 20

μm increments and angle between the blade and the bone was 10°. Next, half of the mouse bone was placed in a transparent plastic mold (37 × 24 × 5 mm, VWR, Radnor, PA, USA) facing flat side down and embedding material of choice was poured on top and around the bone. Embedding material was smoothed out using a stainless steel blade (39.6 × 19.6 mm, Ted Pella Inc., Redding,

CA, USA).

6.2.4 IR-MALDESI MSI

The day before analysis, the IR laser was focused on a laser burn paper (ZFC-23, ZAP-IT,

Concord, NH, USA). Estimated size of a spot ablated from burn paper at room temperature was

200 × 200 μm (Figure E.1). Prior to IR-MALDESI imaging, thickness of the frozen Plaster of

Paris cube (refers to the half bone embedded in Plaster of Paris) was measured using digital caliper whose lower jaws were chilled in a dry ice for 1–2 minutes to prevent Plaster of Paris cube from melting. The IR-MALDESI stage was then lowered by the measured value to ensure that the ROI laid precisely at the laser focus. Next, the IR-MALDESI target plate was cooled to −9 °C, and the relative humidity (RH) in the enclosure box (box around IR-MALDESI source) was lowered to

12%. The frozen Plaster of Paris cube was placed on top of a pre-cooled IR-MALDESI stage, while RH was maintained at 12% to prevent additional ice formation on top of the bone. The sample was equilibrated for 10 minutes to reach stable temperature. A mid-IR laser pulse (IR-

Opolette 2371, Opotek, Carlsbad, CA, USA) with a wavelength of 2940 nm, frequency of 20 Hz, and pulse width of 7–9 ns was used to desorb primarily neutrals from each voxel on the bone. An

105 incident energy of 1.2 mJ per pulse was measured using a pyroelectric detector (Nova 2, Ophir,

Jerusalem, Israel). Spot-to-spot spacing and ROI were 150 μm and 109 × 30 voxels, respectively.

All mass spectrometry measurements were performed on a Q Exactive Plus (Thermo Fisher

Scientific, Bremen, Germany) mass spectrometer. Data were collected across m/z 250–1000 in positive ESI mode using 0.2% formic acid (Sigma-Aldrich, St. Louis, MO, USA) in 50 : 50 (v/v) methanol: water as solvent. Methanol was purchased from Burdick and Jackson (Muskegon, MI,

USA). The ESI flow rate was 1.5 μL min−1, spray voltage was 3.8 kV, MS inlet capillary temperature was 350 °C, and S-lens RF level was 50%. The automatic gain control (AGC) target was disabled due to the pulsed nature of IR-MALDESI source and injection time (IT) was set to

25 ms. Polysiloxane [M + H+]+ ion at m/z 371.1012 was used as a lock mass.

6.2.5 Optical Images

Optical images of bones not embedded in Plaster of Paris (Figures 6.1 and 6.2) and ablations spots on burn paper (Figure E.1) were acquired using a 5× objective on a laser microdissection microscope LMD7000 (Leica, Buffalo Grove, IL, USA). Optical images of bones embedded in Plaster of Paris (Figure 6.6A) were collected using a charge-coupled device (CCD) camera (Point Grey Research Chameleon USB 2.0) positioned right above the Peltier-cooled IR-

MALDESI stage.

6.2.6 Data Analysis

Features were putatively annotated using METASPACE39 annotation engine, and

MSiReader40 imaging software was used to generate selected ion heatmaps. The .RAW files generated by Q Exactive Plus were processed in XCalibur (version 2.2, Thermo Fisher Scientific,

San Jose, CA, USA) and converted into the .mzML format using the open-source MSConvertGUI tool from ProteoWizard41 followed by conversion into .imzML files using .imzML converter.42

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Figure 6.1. Optical image of a 50 μm-thick mouse tibia embedded in carboxymethyl cellulose (CMC)/gelatin medium. The bone was sectioned at −20 °C using Leica CM 1950 cryomicrotome with a Surgipath high-profile disposable blade. Scale bar is 2.5 mm.

Figure 6.2. Optical image of mouse tibia cut in half (top view). Scale bar is 2.5 mm.

6.2.7 Embedding Materials other than Plaster of Paris

CMC/gelatin mixture was prepared by mixing 1 g of CMC (carboxymethyl cellulose sodium salt, medium viscosity, Sigma-Aldrich, St. Louis, MO, USA) with 2 g of gelatin (gelatin from porcine skin, powder, gel strength ∼300 g Bloom, type A, Sigma-Aldrich, St. Louis, MO,

USA) in 20 mL of Optima™ LC/MS grade water. The mixture was stirred with a sterile spatula and heated in a microwave (for less than 30 seconds). The mixture was cooled at room temperature

107 prior to embedding.

Agarose mixture was prepared by mixing 0.3 g of agarose (agarose LE, ultra-pure molecular biology grade, Biotium, Inc., Fremont, CA, USA) with 30 mL of Optima™ LC/MS grade water.

Liquid Nails® (Fuze It, gray, all surface construction adhesive) and thin-set mortar (Versa

Bond, gray, fortified thin-set mortar) were purchased from Home Depot.

Alginate (CAVEX color change alginate, fast set, dust free) was purchased from Amazon.

6.2.8 Data Availability

Putative annotations and corresponding heatmaps are available to the public at https://metaspace2020.eu/project/Khodjaniyazova-2019-bones. Additional data that support the findings of this study are available from the corresponding author upon request.

6.3 Results and Discussion

6.3.1 Laser Focus in IR-MALDESI Imaging

We sought to develop a straightforward and reproducible sample preparation method for direct analysis of bones using IR-MALDESI MSI. Our main goal was to avoid significant tissue- processing steps, such as decalcification, where various solvents are diffused into every part of the tissue, causing chemical and physical changes. IR-MALDESI is a laser sampling technique that requires each spot within the region of interest (ROI) to be positioned at exactly the same distance from the laser focus to ensure uniform sampling. Sample located anywhere outside the depth of focus results in decreased ion signal or no signal at all. Laser focus is never a problem when imaging sections with uniform thickness, because prior to imaging stage height can be adjusted so that laser focus is at the sample surface. Therefore, the first step in method development was to obtain bone section with a uniform thickness.

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6.3.2 Challenges of Bone Imaging using IR-MALDESI

Prior to IR-MALDESI imaging of soft tissues we use a cryomicrotome to slice frozen samples in sections with a thickness between 5 and 200 μm. The frozen slice is easily adhered to a microscope slide that has a temperature slightly higher than that of a cryostat chamber. However, cutting unmodified mouse bone using a cryomicrotome is challenging, because upon slicing all parts of the bone cannot be transferred to a microscope slide (Figure 6.1). In fact, the bone slice tends to curl and does not adhere to a microscope slide as easily as soft tissue. This is due to the complex composition of a bone, which consists of both hard-calcified bone tissue and soft marrow.

Therefore, decalcification and embedding in a material with similar stiffness is the most common practice that facilitates bone sectioning.

An alternative to imaging thin bone sections is imaging half of a bone longitudinally so that less tissue is lost (Figure 6.2). The main difficulty with analyzing half of a bone is the fact that the flat surface of the bone is not parallel to the IR-MALDESI stage due to a slight tilt from metaphyses (Figure 6.3). Therefore, the distance between each sampled spot and laser focus will vary across the bone resulting in a non-uniform sampling. Also, half of a bone positioned on the mobile stage is not as stable as a thin bone section, and it is harder to keep bone tissue frozen, because only a reduced area comes in contact with the Peltier-cooled IR-MALDESI stage. The problem with the tilt (Figure 6.3) can be solved by filling the space between the stage and the bone with material that would level the flat surface of the bone (Figure 6.4F). We report sample preparation steps for longitudinal analysis of native bones embedded in Plaster of Paris, a material compatible with IR-MALDESI MSI (Figure 6.4). The rationale for Plaster of Paris as the embedding material of choice is discussed in the Criteria for embedding material section.

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Figure 6.3. Schematic (side view) illustrating how half of a bone would be positioned on the IR- MALDESI stage and under the IR laser.

6.3.3 Embedding Protocol for IR-MALDESI MSI of Mouse Bones

(1) Rapidly dissect bone using sterile tools immediately following sacrifice. Flash-freeze the bone to prevent morphological changes (Figure 6.4A).

(2) Using cryomicrotome, trim bone at −20 °C until roughly half of the bone is cut away

(Figure 6.4B). For more details about how to cut bone in half see Cryosectioning section in the

Experimental.

(3) Equilibrate bone (cut in half) and pre-cleaned plastic, transparent mold (37 × 24 × 5 mm, VWR, Radnor, PA, USA) for 5–10 minutes at −20 °C inside cryomicrotome chamber. After equilibration, place bone cut in half in the mold with the flat side facing down. Make sure that the flat side of the bone is touching the mold uniformly (Figure 6.4C). Use just enough pressure to hold the bone section flat against the mold surface. Transfer the plastic mold with the bone in it to the cryomicrotome chamber and wait until bone marrow freezes to the mold.

(4) In the cryomicrotome chamber, carefully pour Plaster of Paris (2 : 1 Plaster : water, volume : volume) around the bone. Wait until the Plaster of Paris freezes (Figure 6.4D).

(5) In the cryomicrotome chamber, carefully pour fresh Plaster of Paris until the bone is fully covered in Plaster. Use pre-cleaned blade to smooth out the Plaster of Paris (Figure 6.4E).

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Figure 6.4. Proposed protocol for bone embedding. (A) Flash-freeze fresh bone; (B) cut bone in half using cryomicrotome (see Cryosectioning section in the Experimental for more details); (C) place trimmed bone in a mold facing flat side down; (D) affix bone to the mold using embedding material and wait until the material sets; (E) pour the rest of the embedding material and smooth out the top surface with a blade. (F) and (G) show ROI and direction of laser sampling, respectively.

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(6) After the Plaster of Paris has frozen, remove the Plaster block from the plastic mold and place the block on a pre-cooled target plate (t < 273 K) (Figure 6.4F). To prevent the Plaster block from sliding on the metallic target plate, carefully pour OCT around the Plaster block while avoiding the ROI (Figure 6.4G). Let the OCT freeze and proceed with MSI.

6.3.4 Criteria for Embedding Material

During method development, we created a list with properties that the embedding material should have to be compatible with IR-MALDESI imaging on bones:

(1) Embedding material should not be ‘reactive’ with bone to maximally preserve its initial

(i.e., physiological) composition. This means that the embedding material should not penetrate the outer bone cortex. Also, after pouring/applying the embedding material on top of the bone sectioned in half (Figures 6.4D and E), embedding material should have a high enough viscosity to not leak under the bone and directly contact the marrow.

(2) Embedding material should have a freezing point below 273 K, because during IR-

MALDESI imaging, the target plate is kept below 273 K.

(3) Embedding material should not suppress ESI signal, because IR-MALDESI data are collected from rectangular ROIs (Figure 6.4G) that include both on-tissue (bone) and off-tissue

(embedding material) regions. Therefore, unstable signal collected from the off-tissue region might affect the quality and authenticity of on-tissue spectra.

We tested seven embedding materials with variable physical states (Table 6.1):

CMC/gelatin medium, water, agarose, Liquid Nails®, thin-set mortar, dental alginate, and Plaster of Paris. Following steps listed in Figure 6.4, fresh mouse bones were embedded in each of the seven materials; however, only Plaster of Paris satisfied all three requirements (Table 6.1): Plaster of Paris can be easily distributed around the bone without leaking underneath the bone section

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(Figure 6.5A); the abundance of biomolecules is lower in off-tissue regions compared to on-tissue regions (Figure 6.5B); and spectra collected from off-tissue (Figure 6.5C) and on-tissue (Figure

6.5D) regions have stable signal with acceptable signal-to-noise (S/N) ratios. The other six embedding materials (CMC/gelatin, water, agarose, Liquid Nails, thin-set mortar, and alginate) are not compatible with the reported method because they either modify bone, do not freeze at 273

K, or suppress signal of bone-specific ions. For example, CMC/gelatin and agarose media have to be poured into a mold while still being warm (t > 298 K) because CMC/gelatin and agarose mixtures congeal at lower temperatures which make even spreading around the bone almost impossible. Moreover, heat stress (e.g., warm embedding material around the bone) affects composition and dynamics of the lipidome.43,44 Water and agarose, unlike CMC/gelatin, Liquid

Nails, thin-set mortar, alginate, and Plaster of Paris, easily leak under the bone during embedding process (Figures 6.4D and E) and therefore come in direct contact with the surface of the bone to be sampled with IR laser (e.g., ROI). We hypothesize that direct contact of water or agarose with frozen bone might affect the spatial distribution of bone-specific features.

Table 6.1. List of embedding materials used for IR-MALDESI MSI of mouse bones.

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Figure 6.5. IR-MALDESI MSI of a stroke-affected mouse humerus embedded in Plaster of Paris. (A) optical image (+40% brightness, +40% contrast); (B) unnormalized and not background- subtracted cholesterol ion heatmap; MS1 spectra collected from (C) off-tissue and (d) on-tissue regions. Scale bar is 1.5 mm.

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To demonstrate the feasibility of IR-MALDESI for 2D MSI of bones, humeri derived from healthy and stroke-affected mice were embedded in Plaster of Paris following the protocol from Figure 6.4 and analyzed using IR-MALDESI coupled with a Q Exactive Plus. Optical images and ion heatmaps with putatively annotated features are shown in Figures 6.6A and B–J, respectively. Ion heatmaps in Figure 6.6 demonstrate that IR-MALDESI is capable of detecting features unique not only to the entire humerus (C, D and F) but also to its parts: cortical bone (b, e and h), marrow tissue (J), and trabecular (e.g., cancellous) bone (G and I). We do not report unique identifications (IDs) based on MS/MS because this work is technical. However, future work will be focused on detailed characterization of bone tissues using MS/MS, comparison between bones from healthy and stroke-affected mice as well as bones from paretic and non-paretic limbs from stroke-affected mice. Overall, 826 and 669 tissue-specific features (Tables E.1 and E.2) were putatively annotated in healthy and stroke-affected bones, respectively, using

METASPACE39 annotation engine (https://metaspace2020.eu/project/Khodjaniyazova-2019- bones).

We developed a protocol for a direct analysis and MSI of fresh, undecalcified mouse bones using IR-MALDESI MSI to avoid excessive and time-consuming sample preparation steps that modify the bone. Our method allows to detect a broader range of endogenous biomolecules, because prior to imaging, bone does not undergo any internal chemical treatments, such as decalcification, where bone is soaked in various solvents for days and sometimes even weeks. In this method, the bone was cut in half longitudinally and embedded in Plaster of Paris so that all parts of the bone except for the cut surface came in contact with the Plaster. Plaster of Paris helps to position the cut of the bone surface parallel to the target plate and is compatible with IR-

MALDESI MSI.

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Figure 6.6. IR-MALDESI MSI of healthy and stroke-affected mouse humeri embedded in Plaster of Paris. (a) optical image (+40% brightness, +40% contrast) and (b–j) ion heatmaps. Features from MS1 scans were putatively annotated using METASPACE annotation engine. Scale bar is 1.5 mm.

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To demonstrate the feasibility of our reported method, healthy and stroke-affected mouse humeri were embedded in Plaster of Paris and imaged using IR-MALDESI source. METASPACE was used to putatively annotate 826 and 669 tissue-specific features in healthy and stroke-affected humeri, respectively. Generated heatmaps show features specific not only to the whole bone but also to its separate parts such as cortical bone, marrow tissue, and trabecular bone.

Besides embedding, we also tried sectioning mouse bone using a Techcut 4™ low-speed saw (Allied High Tech Products Inc., Rancho Dominguez, CA, USA) with a diamond-coated blade to obtain a 500 to 1000 μm-thick bone section with a uniform thickness. A whole mouse femur was embedded in each of the seven embedding materials listed in Table 6.1 and clamped above the diamond-coated blade. During sectioning, the diamond-coated blade had to be constantly irrigated with cold water to prevent sample and blade from heating. As a result of this constant contact with the water, each of the seven embedding materials around the bone softened and eventually fell apart.

For bones and other sample types where control of the area around the sample (i.e., embedding material) is required, the IR-MALDESI platform has been modified for imaging an arbitrarily shaped (non-rectangular) ROI [Garrard, K. P., Ekelöf, M., Khodjaniyazova, S., Bagley,

M. C., and Muddiman, D. C. RastirX: a Versatile Platform for Imaging Arbitrary Spatial Patterns.

Manuscript in preparation]. By controlling the number of off-tissue pixels sampled, alginate could potentially be used as an embedding material, as well (Table 6.1).

6.4 Conclusions

A method utilizing IR-MALDESI ionization source coupled with a Q Exactive Plus mass spectrometer was developed as a facile and rapid method for direct analysis of mouse bones. To ensure uniform sampling, half of the bone was embedded in Plaster of Paris which was 1 of 7

117 tested materials that satisfied requirements for IR-MALDESI MSI. Future work will be focused on finding biomarkers specific to disease-affected bones

6.5 Acknowledgements

The authors gratefully acknowledge the financial support received from National Institute of Health (NIH, R01GM087964), American Heart Association (AHA, 7GRNT33710007),

American Society of Biomechanics, and North Carolina State University (NCSU). The authors also thank the Analytical Instrumentation Facility (AIF) at NCSU (ECCS-1542015) for providing access to the low-speed saw. All mass spectrometry measurements were made in the Molecular

Education, Technology and Research Innovation Center (METRIC) at NCSU.

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6.6 References

1. F. Leung, L. S. Eberlin, K. Schwamborn, R. M. Heeren, N. Winograd and R. G. Cooks, Clin. Chem., 2019, 65, 510–513.

2. L. S. Eberlin, A. L. Dill, A. B. Costa, D. R. Ifa, L. Cheng, T. Masterson, M. Koch, T. L. Ratliff and R. G. Cooks, Anal. Chem., 2010, 82, 3430–3434.

3. M. Stoeckli, P. Chaurand, D. E. Hallahan and R. M. Caprioli, Nat. Med., 2001, 7, 493.

4. L. A. McDonnell and R. M. Heeren, Mass Spectrom. Rev., 2007, 26, 606–643.

5. R. M. Caprioli, T. B. Farmer and J. Gile, Anal. Chem., 1997, 69, 4751–4760.

6. S. R. Ellis, A. L. Bruinen and R. M. Heeren, Anal. Bioanal. Chem., 2014, 406, 1275– 1289.

7. M. T. Bokhart, E. Rosen, C. Thompson, C. Sykes, D. Kashuba and D. C. Muddiman, Anal. Bioanal. Chem., 2015, 407, 2073–2084.

8. M. Bokhart and D. Muddiman, Analyst, 2016, 141, 5236–5245.

9. M. Nazari, M. T. Bokhart, P. L. Loziuk and D. C. Muddiman, Analyst, 2018, 143, 654– 661.

10. T. Goldschlager, A. Abdelkader, J. Kerr, I. Boundy and G. Jenkin, J. Visualized Exp., 2010, e1707.

11. K. Schaepe, D. R. Bhandari, J. Werner, A. Henss, A. Pirkl, M. Kleine-Boymann, M. Rohnke, S. Wenisch, E. Neumann and J. r. Janek, Anal. Chem., 2018, 90, 8856–8864.

12. Henss, A. Hild, M. Rohnke, S. Wenisch and J. Janek, Biointerphases, 2016, 11, 02A302.

13. P. Malmberg and H. Nygren, Proteomics, 2008, 8, 3755–3762.

14. B. Rocha, C. Ruiz-Romero and F. J. Blanco, Nat. Rev. Rheumatol., 2017, 13, 52.

15. T. B. Bemenderfer, J. S. Harris, K. W. Condon and M. A. Kacena, in Skeletal Development and Repair, Springer, 2014, pp. 123–147.

16. Porter, R. Irwin, J. Miller, D. J. Horan, A. G. Robling and L. R. McCabe, Sci. Rep., 2017, 7, 42505.

17. S. D. Stout and S. L. Teitelbaum, Am. J. Phys. Anthropol., 1976, 44, 263–269.

18. G. J. Maat, R. P. Van Den Bos and M. Aarents, Int. J. Osteoarchaeol., 2001, 11, 366–374.

19. L. Zhang, B. L. Tai, G. Wang, K. Zhang, S. Sullivan and J. Shih, Med. Eng. Phys., 2013, 35, 1391–1398.

119

20. B. L. Tai, L. Zhang, A. Wang, S. Sullivan and A. J. Shih, Procedia CIRP, 2013, 5, 226– 230.

21. G. Robichaud, J. A. Barry and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2014, 25, 319–328.

22. J. A. Barry, G. Robichaud, M. T. Bokhart, C. Thompson, C. Sykes, A. D. Kashuba and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2014, 25, 2038–2047.

23. M. Ekel¨of, E. K. McMurtrie, M. Nazari, S. D. Johanningsmeier and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2017, 28, 370–375.

24. C. G. Thompson, M. T. Bokhart, C. Sykes, L. Adamson, Y. Fedoriw, P. A. Luciw, D. C. Muddiman, D. M. Kashuba and E. P. Rosen, Antimicrob. Agents Chemother., 2015, 59(5), 2944–2948.

25. M. Nazari, A. A. Malico, M. Ekel¨of, S. Lund, G. J. Williams and D. C. Muddiman, Anal. Bioanal. Chem., 2018, 410, 953–962.

26. C. Thompson, E. Rosen, M. Bokhart, H. Prince, C. Sykes, D. C. Muddiman and A. D. Kashuba, AIDS Res. Hum. Retroviruses, 2014, 30, A69.

27. E. P. Rosen, C. G. Thompson, M. T. Bokhart, H. M. Prince, C. Sykes, D. C. Muddiman and A. D. Kashuba, Anal. Chem., 2015, 88, 1336–1344.

28. K. H. Cochran, J. A. Barry, G. Robichaud and D. C. Muddiman, Anal. Bioanal. Chem., 2015, 407, 813–820.

29. K. H. Cochran, J. A. Barry, D. C. Muddiman and D. Hinks, Anal. Chem., 2012, 85, 831– 836.

30. M. Nazari, M. T. Bokhart and D. C. Muddiman, J. Visualized Exp., 2016, e53942.

31. J. S. Sampson, A. M. Hawkridge and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2006, 17, 1712–1716.

32. M. Nazari, M. Ekel¨of, S. Khodjaniyazova, N. L. Elsen, J. D. Williams and D. C. Muddiman, Rapid Commun. Mass Spectrom., 2017, 31, 1868–1874.

33. L. Boskey, BoneKEy Rep., 2013, 2, 447.

34. R. B. Dixon and D. C. Muddiman, Analyst, 2010, 135, 880–882.

35. Tu and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2019, 1–12.

36. E. Z. Longa, P. R. Weinstein, S. Carlson and R. Cummins, Stroke, 1989, 20, 84–91.

37. H. Sakai, H. Sheng, R. B. Yates, K. Ishida, R. D. Pearlstein and D. S. Warner, Anesthesiology, 2007, 106, 92–99.

120

38. N. J. Hanne, A. J. Steward, M. R. Sessions, H. L. Thornburg, H. Sheng and J. H. Cole, BioRxiv, 2019, 708388.

39. Palmer, P. Phapale, I. Chernyavsky, R. Lavigne, D. Fay, Tarasov, V. Kovalev, J. Fuchser, S. Nikolenko, C. Pineau, M. Becker and T. Alexandrov, Nat. Methods, 2016, 14, 57.

40. M. T. Bokhart, M. Nazari, K. P. Garrard and D. C. Muddiman, J. Am. Soc. Mass Spectrom., 2018, 29, 8–16.

41. D. Kessner, M. Chambers, R. Burke, D. Agus and P. Mallick, Bioinformatics, 2008, 24, 2534–2536.

42. M. Race, I. B. Styles and J. Bunch, J. Proteomics, 2012, 75, 5111–5112.

43. G. Balogh , M. Péter , A. Glatz , I. Gombos , Z. Török , I. Horváth , J. L. Harwood and L. Vígh, FEBS Lett., 2013, 587, 1970–1980.

44. S. L. Neidleman, Biotechnol. Genet. Eng. Rev., 1987, 5, 245–268.

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APPENDICES

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Appendix A

Supplemental Information for Chapter 2

Figure A.1. Full range MS spectrum of homemade calibration mixture. This spectrum represents the average of ninety-nine transient scans at AGC target of 1 × 106.

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Figure A.2. Distributions of mass measurement accuracy (MMA) across different ion populations for (A) caffeineSigma, (B) MRFA, and (C) ultramark 1421. Theoretical masses were obtained from IUPAC. Each data point represents a single transient scan.

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13 15 13 34 Figure A.3. Calculated δOrbitrap for (A, B) C and N in caffeineSigma, (C, D) C and S in MRFA, and (E) 13C in ultramark 1421 across eight AGC targets. Each data point represents a single transient scan.

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13 Figure A.4. Relative abundance of C in caffeineSigma, MRFA, and ultramark 1421 are shown in red, blue, and green, respectively. All three atom percent values fall in the range of 13C abundances found in nature (black dashed lines); however, they are clearly different than the “best measurement” value reported by IUPAC.

Figure A.5. Orbitrap’s deviations from the true number of (A) nitrogens in caffeineSigma and (B) sulfurs in MRFA across different AGC targets. Atom percent values obtained from IRMS analyses (circles) and also from IUPAC (triangles) are plotted together for simple comparison. Error bars represent 95% confidence intervals for n=99.

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13 Figure A.6. Difference between expected and observed relative abundances of C2 peak in 13 MRFA peptide. Expected relative abundances of C2 peak were calculated using probability Equation 2.4.

Figure A.7. Deviations from the known number of carbons across different ion populations. Atom percent values, used in carbon counting, were obtained from IUPAC. Error bars correspond to 95% confidence interval (n=99).

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Appendix B

Supplemental Information for Chapter 3

Figure B.1. Distribution of (A) grades, (B) MMAs, and (C) m-scores for lipid ions detected by shotgun and IR-MALDESI.

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Figure B.2. (A) Searching all lipid ions detected via shotgun against IR-MALDESI raw data. (B) Searching all lipid ions detected via IR-MALDESI against shotgun raw data. MS1 scan numbers are shown on the x-axis and monoisotopic ion abundances are on the y-axis. Each line represents lipid ion.

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Table B.1. One hundred most abundant background ions detected in a shotgun (2nd column) and IR-MALDESI (3rd column) analyses across four m/z ranges: m/z 150-600 positive ESI, m/z 500- 2000 positive ESI, m/z 150-600 negative ESI, and m/z 500-2000 positive ESI.

background ions shotgun IR-MALDESI 387.1792 300.2778 387.1801 300.2784 387.1783 300.2772 387.1810 371.1016 387.1774 371.1007 387.1819 371.1024 388.1830 284.3314 388.1821 300.2790 388.1839 284.3308 404.2062 300.2765 404.2052 371.0999 388.1812 300.7795 404.2072 284.3319 387.1765 284.3302 387.1828 198.2094 388.1849 300.7801 404.2043 198.2091 150-600, positive ESI 540.4244 371.1033 540.4259 300.7788 267.1225 387.1802 202.1801 195.1017 267.1220 387.1811 202.1797 198.2098 283.1169 387.1793 316.2114 300.2796 404.2081 195.1021 409.1613 198.2088 283.1175 371.0990 315.2528 313.1433 568.4567 300.7807 315.2521 195.1014 387.1918 284.3325 388.1803 313.1439 316.2108 300.7782

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387.1909 300.2759 568.4551 313.1426 409.1623 284.3297 540.4273 372.1006 540.4229 195.1024 267.1230 387.1820 409.1603 372.0997 177.0069 230.2479 316.2121 387.1784 202.1804 372.1014 283.1164 404.2063 279.0929 404.2073 267.1214 177.9925 202.1794 373.0985 280.1359 230.2483 568.4583 198.2101 387.1927 371.1041 315.2534 163.9769 280.1364 163.9767 195.1378 373.0976 279.0924 313.1446 257.2472 279.1592 177.0066 198.2084 283.1181 177.9928 177.0072 177.9922 257.2467 230.2474 387.1900 195.1011 255.1585 285.3347 315.2514 285.3341 208.1542 373.0993 255.0831 393.2856 279.0935 313.1419 255.0836 279.1587 271.2626 205.9873 195.1381 355.0700 255.1590 191.9719 405.2093 404.2083 208.1539 393.2865 259.1898 372.1023

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285.2782 228.1959 489.2261 327.0780 568.4535 301.2849 316.2101 404.2054 487.3261 205.9877 404.2033 338.3417 158.9963 372.0989 195.1374 217.1073 373.2582 198.7111 280.1353 376.2591 363.2369 257.2476 259.1903 198.7107 373.2574 195.1231 489.2274 279.1598 409.1633 195.1227 388.1858 163.9772 285.2787 374.2111 409.1594 191.9716 487.3248 355.0692 257.2477 230.2487 271.2632 376.2600 271.2621 217.1069 325.3098 327.0787 405.2103 300.7813 363.2377 163.9764 255.1580 374.2119 158.9966 228.1963 255.2329 255.2335 255.2334 255.2330 255.2325 255.2339 283.2646 283.2647 283.2641 283.2652 255.2339 255.2325 150-600, negative ESI 283.2652 283.2641 255.2320 255.2344 283.2635 283.2658 283.2658 311.1692 255.2344 325.1847 312.9190 283.2635

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312.9184 311.1699 256.2364 325.1854 312.9197 255.2320 283.2629 311.1686 256.2359 325.1840 255.2315 283.2664 256.2369 256.2369 284.2680 256.2364 284.2674 311.1705 211.1336 325.1861 312.9177 255.2349 211.1333 265.1483 312.9204 207.9311 256.2354 284.2680 283.2663 297.1536 284.2686 265.1488 284.2669 311.1679 211.1340 207.9307 255.2349 256.2374 256.2374 256.2359 253.1448 297.1530 197.8072 284.2686 211.1329 325.1833 445.1874 284.2675 197.8076 207.9314 253.1443 297.1543 445.1863 265.1478 253.1453 195.0314 195.8103 283.2630 157.1224 157.1228 283.2624 157.1225 445.1885 265.1493 157.1226 195.0318 197.8069 243.8995 312.9171 171.1384 256.2349 325.1868 199.1699 207.9304 284.2691 195.0311 195.8100 311.1712

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195.8106 297.1524 197.8079 284.2692 312.9210 243.9000 211.1344 255.2315 255.2310 171.1021 199.1695 293.1798 284.2663 171.1387 241.2174 242.1764 241.2170 171.1382 157.1221 256.2379 421.1560 157.1230 421.1550 269.2127 205.1594 256.2354 445.1852 447.1357 253.1438 243.8991 199.8044 284.2669 205.1590 157.1223 170.8325 293.1804 253.1457 297.1549 211.1326 207.9318 170.8322 171.1023 157.1228 221.9470 525.9786 312.1728 199.1702 447.1346 227.2016 165.0207 525.9772 283.2669 227.2012 326.1884 283.2669 179.9359 445.1897 242.1768 199.8047 171.1018 256.2378 278.8538 159.0652 165.0204 159.0655 265.1473 209.1543 269.2132 199.8041 293.1793 241.2179 221.9466 199.1692 179.9356 421.1571 242.1759 205.1597 195.0321

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241.2165 255.2354 195.8096 220.1468 421.1427 157.0863 195.8109 312.1721 421.1540 326.1877 197.8066 243.9004 421.1437 227.2016 209.1540 339.2007 171.1382 241.2174 170.8328 278.8544 637.3062 637.3054 637.3043 637.3073 537.8791 637.3092 637.3081 637.3035 537.8777 654.3332 537.8806 654.3312 637.3024 654.3352 537.8821 638.3101 537.8762 519.1400 637.3100 637.3111 555.8889 637.3016 555.8904 638.3082 638.3090 654.3293 638.3071 519.1385 500-2000, positive ESI 638.3109 519.1414 637.3005 638.3120 555.8920 654.3372 533.8852 655.3367 555.8873 638.3063 533.8837 655.3348 533.8867 536.1666 540.4267 519.1371 540.4252 519.1428 638.3052 655.3387 537.8836 536.1651 638.3128 638.3140 537.8747 593.1588 535.8832 654.3273 568.4573 520.1401

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535.8847 536.1681 538.8820 520.1387 637.3119 599.5485 533.8823 593.1571 568.4557 637.3130 538.8806 599.5502 555.8858 593.1605 555.8936 655.3328 540.4281 659.2876 540.4237 610.1855 568.4589 637.2997 533.8881 520.1415 538.8835 654.3392 1273.6043 659.2896 535.8817 536.1637 535.8862 638.3044 1273.6097 610.1837 565.9104 655.3407 565.9120 520.1373 538.8791 599.5468 637.2986 521.1361 1273.5989 610.1873 568.4541 599.5520 1274.6074 659.2856 638.3033 536.1695 553.8940 521.1375 1274.6128 519.1357 568.4605 593.1553 638.3147 519.1442 556.8927 639.3134 553.8955 593.1622 639.3104 639.3115 540.4296 594.1588 639.3123 659.2916 538.8850 521.1347 541.4292 520.1429 540.4222 537.1662 556.8912 503.1083 1273.6151 638.3159

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565.9088 667.1767 545.2364 610.1819 565.9136 594.1571 533.8808 537.1647 545.2349 594.1605 535.8803 667.1788 1274.6020 503.1097 553.8924 655.3308 534.8857 537.1677 534.8872 639.3153 541.4307 521.1389 569.4599 611.1859 535.8876 768.2504 556.8943 610.1890 569.4615 611.1841 541.4277 639.3096 537.8851 600.5525 551.8944 503.1070 551.8960 520.1359 533.8896 599.5450 537.8732 607.2114 1274.6182 659.2836 555.8951 684.2043 1273.5935 667.1747 639.3085 768.2479 531.8896 607.2096 639.3142 654.3253 555.8842 536.1622 553.8971 656.3386 663.4535 600.5542 654.3321 599.5537 556.8896 624.2367 695.3129 681.2977 695.3151 681.2998 695.3108 681.2956 500-2000, negative ESI 525.9778 681.3019 525.9792 681.2935 671.2680 682.3026 671.2701 682.3005

137

695.3173 695.3130 525.9763 695.3152 695.3086 681.3040 525.9806 682.3048 671.2659 682.2984 671.2721 695.3109 696.3164 695.3174 891.5333 681.2914 696.3185 698.2883 696.3142 698.2861 891.5301 682.3069 891.5364 698.2905 525.9749 682.2963 641.4064 695.3087 641.4084 696.3165 919.5632 696.3187 695.3195 695.3196 919.5665 696.3143 525.9821 698.2839 672.2715 683.3056 671.2639 671.2702 672.2736 683.3035 671.2742 696.3208 696.3207 681.3061 891.5270 653.3033 892.5348 698.2927 695.3064 671.2681 673.2648 653.3053 641.4045 653.3014 919.5599 699.2916 892.5380 671.2722 696.3120 683.3013 641.4103 683.3077 669.4382 891.5334 672.2694 681.2893 891.5396 699.2938 673.2628 699.2894 673.2669 671.2661 669.4402 695.3065

138

919.5698 696.3121 672.2756 682.2942 920.5660 891.5366 892.5317 891.5303 920.5694 682.3090 892.5412 697.2937 698.2882 641.4085 669.4361 696.3230 642.4113 698.2818 642.4094 653.2994 698.2860 653.3073 919.5566 641.4065 525.9735 697.2915 920.5627 698.2949 696.3229 725.3258 669.4423 725.3235 698.2904 697.2959 641.4026 671.2743 673.2690 683.2992 920.5727 699.2960 697.3198 641.4104 641.4122 892.5382 673.2607 695.3217 891.5238 683.3098 642.4132 699.2872 526.9820 919.5634 672.2674 892.5350 697.3176 919.5667 671.2618 672.2737 696.3098 654.3065 635.2918 891.5398 695.3216 697.3199 672.2777 891.5271 670.4417 672.2716 697.3220 725.3282 521.2900 641.4046 674.2687 725.3212 892.5285 639.2870 631.4224 681.3082

139

919.5731 697.2893 771.3447 654.3085 526.9806 697.3221 642.4074 671.2640 641.4199 673.2670 525.9835 654.3045 553.2801 697.3177 698.2838 639.2889 635.2937 892.5413 521.2886 673.2649 671.2763 669.4383 657.4018 669.4404 631.4243 672.2757 670.4438 919.5601 526.9835 681.2872

Table B.2. Lipid classes that can be annotated by LipidSearchTM software. lipid group lipid class lipid name CL cardiolipin DLCL cardiolipin MLCL cardiolipin LPA lysophosphatidic acid PA phosphatidic acid LPC lysophosphatidylcholine PC phosphatidylcholine LPE lysophosphatidylethanolamine PE phosphatidylethanolamine phospholipids LPG lysophosphatidylglycerol PG phosphatidylglycerol LPI lysophosphatidylinositol PI phosphatidylinositol LPS lysophosphatidylserine PS phosphatidylserine PIP phosphatidylinositol PIP2 phosphatidylinositol PIP3 phosphatidylinositol Cer ceramides CerP ceramides phosphate sphingolipids CerPE ceramide phosphoethanolamines Hex1SPH glucosylsphingosine Hex1Cer simple Glc series

140

Hex2Cer simple Glc series Hex3Cer simple Glc series CerG2GNAc1 simple Glc series CerG3GNAc1 simple Glc series CerG3GNAc2 simple Glc series GM3 gangliosides GM2 gangliosides GM1 gangliosides GD1a gangliosides GD1b gangliosides GD2 gangliosides GD3 gangliosides GT1a gangliosides GT1b gangliosides GT1c gangliosides GT2 gangliosides GT3 gangliosides GQ1c gangliosides GQ1b gangliosides LSM lysosphingomyelin phSM sphingomyelin(phytosphingosine) SM sphingomyelin SPH sphingosine SPHP sphingosine phosphate ST sulfatide AcHexSiE acylGlcSitosterol ester AcHexStE acylGlcStigmasterol ester AcHexZyE acylGlcZymosterol ester AcHexCmE acylGlcCampesterol ester AcHexChE acylGlcCholesterol ester ChE cholesterol ester D7ChE deuterated cholesterol ester CmE campesterol ester neutral lipids DG diglyceride D5DG deuterated diglyceride MG monoglyceride SiE sitosterol ester StE stigmasterol ester TG triglyceride D5TG deuterated triglyceride ZyE zymosterol ester

141

AcCa acyl carnitine AEA N-Acylethanolamine Co coenzyme cPA cyclic phosphatidic acid FA fatty acid LPEt lysophosphatidylethanol fatty acyl and other lipids LPMe lysophosphatidylmethanol OAHFA OAcyl-(gamma-hydroxy)FA PAF platelet-activating factor PEt phosphatidylethanol PMe phosphatidylmethanol SL seminolipid WE wax esters MGMG monogalactosylmonoacylglycerol MGDG monogalactosyldiacylglycerol DGMG digalactosylmonoacylglycerol glycoglycerolipids DGDG digalactosyldiacylglycerol SQMG sulfoquinovosylmonoacylglycerol SQDG sulfoquinovosyldiacylglycerol BiotinylPE glycerophosphoethanolamine-N-(biotinyl) BisMeLPA bis-methyl lysophosphatidic acid BisMePA bis-methyl phosphatidic acid derivatized lipids BisMePE bis-methyl phosphatidylethanolamine (biotinylation, BisMePG bis-methyl phosphatidylglycerol diazomethane) BisMePS bis-methyl phosphatidy lserine MePC methyl phosphatidylcholine dMePE dimethylphosphatidylethanolamine LdMePE lysodimethylphosphatidylethanolamine

Table B.3A. Lipid ions that were confidently detected (based on MS/MS) in shotgun and IR- MALDESI analyses of healthy rat liver. Lipid ions were filtered based on the following thresholds in LipidSearchTM: m-score threshold was set to 5 and ID quality filters were set to include lipids with grades A, B, and C. (A) lipid ions No. only in shotgun only in IR-MALDESI both 1 CerG2(d25:0/20:4)-H DG(16:0/18:2)+NH4 FA(20:4)-H 2 CerG2GNAc1(d22:1/20:1+O)+NH4 DG(16:0/20:4)+H LPC(16:0)+H 3 CerG3(d16:1/22:6)-H DG(16:0/20:4)+NH4 LPC(16:1p)+H 4 CerG3(d17:0/28:4)+NH4 DG(16:1p/18:1)+H PC(16:0/16:1)+H 5 CerG3(d24:0/18:2)-H DG(16:1p/18:2)+H PC(16:0/18:2)+H 6 ChE(20:4)+H DG(16:1p/20:4)+H PE(16:0/20:4)-H

142

7 CL(10:0/20:4/22:6/18:2)-2H DG(18:0/20:4)+NH4 PE(16:0/22:6)-H 8 CL(16:0/18:0/18:0/18:0)-2H DG(18:1/18:1)+NH4 PE(18:0/18:2)-H 9 CL(17:1/18:2/18:2/18:1)-2H DG(18:1/18:2)+NH4 PE(18:0/20:4)-H 10 CL(18:1/16:0/16:0/18:2)-2H DG(18:1/22:4)+NH4 PE(18:0/22:6)-H 11 CL(18:1/16:0/18:1/16:0)-2H DG(18:1p/18:2)+H PE(18:0p/20:4)-H 12 CL(18:1/18:1/18:2/14:0)-2H DG(18:1p/20:4)+H 13 CL(18:2/14:0/18:2/18:1)-H dMePE(16:0/18:2)+H 14 CL(18:2/15:0/18:1/18:2)-2H dMePE(18:0/18:2)+H 15 CL(18:2/16:0/18:0/18:0)-2H dMePE(18:4/18:0)+H 16 CL(18:2/16:0/20:4/20:4)-2H FA(14:1)-H 17 CL(18:2/16:1/16:1/18:2)-H LPC(18:1p)+H 18 CL(18:2/16:1/18:2/18:2)-H PC(15:0/22:6)+H 19 CL(18:2/16:1/18:2/22:6)-2H PC(15:1/22:6)+H 20 CL(18:2/16:1/20:4/16:1)-2H PC(15:1/24:7)+H 21 CL(18:2/18:0/20:4/20:3)-2H PC(16:0/13:0)+H 22 CL(18:2/18:1/18:1/22:6)-2H PC(16:0/18:3)+Na 23 CL(18:2/18:1/18:2/14:0)-2H PC(16:0/22:4)+K 24 CL(18:2/18:1/18:2/16:1)-2H PC(16:0/22:6)+H 25 CL(18:2/18:1/22:6/18:2)-2H PC(16:0/24:7)+H 26 CL(18:2/18:2/15:0/18:2)-2H PC(16:1/16:1)+H 27 CL(18:2/18:2/18:2/18:2)-2H PC(16:1/22:6)+H 28 CL(18:2/18:2/18:2/18:2)-H PC(16:1/24:7)+H 29 CL(18:2/18:2/18:2/20:3)-2H PC(17:0/22:6)+H 30 CL(18:2/18:2/20:3/18:2)-H PC(17:1/22:6)+H 31 CL(18:2/18:2/22:1/18:1)-H PC(17:1/24:7)+H 32 CL(18:2/20:4/18:2/18:2)-H PC(18:0/18:2)+H 33 CL(18:2/22:0/22:4/18:2)-2H PC(18:0/19:4)+H 34 CL(18:2/22:6/18:2/18:2)-H PC(18:0/20:4)+H 35 CL(18:3/16:0/20:4/18:2)-2H PC(18:0/22:6)+H 36 CL(18:3/18:2/18:2/18:2)-H PC(18:1/24:7)+H 37 CL(19:1/18:2/18:2/18:2)-2H PC(18:1p/18:0)+K 38 CL(20:2/18:0/20:4/18:2)-2H PC(18:4/16:0)+H 39 CL(20:4/16:0/20:1/24:1)-2H PC(18:4/18:0)+H 40 CL(20:4/18:1/20:0/24:1)-2H PC(24:5/22:6)+Cl 41 CL(21:0/16:0/16:0/18:2)-2H PE(16:0/18:2)+H 42 CL(21:1/18:1/18:2/18:2)-H PE(16:0/18:2)-H 43 CL(22:1/20:4/16:0/18:2)-H PE(16:0/20:4)+H 44 CL(22:5/18:0/22:5/18:0)-2H PE(16:0/22:6)+H 45 CL(22:6/16:0/18:0/22:6)-2H PEt(16:0/20:3)+K

143

46 CL(23:0/16:0/18:2/22:6)-H PEt(16:1/20:4)-H 47 CL(23:0/18:0/18:0/18:0)-H PEt(18:1/20:4)-H 48 CL(23:1/18:2/16:0/20:4)-H PG(18:0/18:2)+Li 49 CL(23:1/18:2/20:4/18:0)-H PG(18:0/22:6)+NH4 50 CL(23:4/16:1/16:1/16:1)-2H PG(18:3/20:2)+Li 51 CL(24:0/16:0/18:0/16:0)-2H PMe(20:0/13:1)+K 52 CL(24:0/18:0/18:1/18:1)-2H SM(d22:0/32:5)+H 53 CL(24:0/18:0/22:5/22:5)-2H TG(13:1/21:1/21:1)+NH4 54 CL(24:1/20:1/22:6/16:0)-2H TG(16:0/16:0/18:2)+NH4 55 cPA(16:0)-H TG(16:0/16:0/20:3)+K 56 DG(16:0p/16:0)+Na TG(16:0/18:1/18:2)+NH4 57 DG(18:0/20:4)+Na TG(16:0/18:1/22:6)+NH4 58 DGMG(16:1)+HCOO TG(16:0/18:2/18:2)+NH4 59 dMePE(18:0/18:2)-H TG(16:0/18:2/20:3)+NH4 60 dMePE(18:0/20:4)-H TG(16:0/18:2/22:6)+NH4 61 dMePE(18:0/22:6)-H TG(16:2/14:0/22:2)+K 62 FA(20:5)-H TG(16:2/16:2/24:1)+NH4 63 FA(22:6)-H TG(18:0/16:1/22:5)+NH4 64 LPA(16:0)-H TG(18:1/18:1/18:2)+NH4 65 LPC(12:0p)+H TG(18:1/18:1/22:6)+NH4 66 LPC(16:0)+K TG(18:1/18:2/22:6)+NH4 67 LPC(16:0)+Na TG(19:1/16:0/18:2)+NH4 68 LPC(18:0)+H TG(19:1/17:2/19:1)+NH4 69 LPC(18:0)+K TG(20:1/18:3/22:4)+NH4 70 LPC(18:0)+Na ZyE(0:0)+H 71 LPC(18:1)+H 72 LPC(18:2)+K 73 LPC(18:2)+Na 74 LPC(20:4)+H 75 LPC(20:4)+K 76 LPC(20:4)+Na 77 LPC(22:6)+H 78 LPC(22:6)+Na 79 LPE(16:0)-H 80 LPE(17:1)-H 81 LPE(18:0)+Na 82 LPE(18:0)-H 83 LPE(20:4)-H 84 LPE(22:6)-H

144

85 LPG(16:0)-H 86 LPG(18:1)-H 87 LPG(18:2)-H 88 LPG(20:4)-H 89 LPG(22:6)-H 90 LPI(16:0)-H 91 LPI(18:0)-H 92 LPI(20:4)-H 93 LPI(33:0)-H 94 LPI(33:1)-H 95 LPS(18:0)-H 96 LPS(20:4)-H 97 LPS(22:6)-H 98 MG(20:2)+NH4 99 OAHFA(16:0/16:0)-H 100 OAHFA(16:0/17:0)-H 101 OAHFA(16:0/18:2)-H 102 OAHFA(16:0/28:3)-H 103 OAHFA(18:0/16:1)-H 104 OAHFA(18:0/17:0)-H 105 OAHFA(18:0/18:0)-H 106 OAHFA(18:1/18:0)-H 107 OAHFA(18:1/18:1)-H 108 OAHFA(18:2/18:1)-H 109 OAHFA(18:2/20:2)-H 110 OAHFA(18:2/27:1)-H 111 OAHFA(20:4/18:1)-H 112 PA(12:0/18:2)-H 113 PA(12:0/20:4)-H 114 PA(14:0/18:2)-H 115 PA(15:0/18:2)-H 116 PA(16:0p/24:2)+K 117 PA(18:2/22:6)-H 118 PA(18:2p/24:2)+K 119 PA(20:1p/24:2)+Na 120 PC(10:0e/20:4)+Cl 121 PC(12:0e/18:2)+Cl 122 PC(12:0p/14:0)+Cl 123 PC(12:0p/24:7)+Na

145

124 PC(14:0/20:4)+Na 125 PC(14:0p/22:6)+HCOO 126 PC(16:0/16:0)+K 127 PC(16:0/16:0)+Na 128 PC(16:0/16:1)+K 129 PC(16:0/16:1)+Na 130 PC(16:0/18:2)+K 131 PC(16:0/18:2)+Na 132 PC(16:0/18:3)+K 133 PC(16:0/20:4)+K 134 PC(16:0/20:4)+Na 135 PC(16:0/22:6)+K 136 PC(16:0/22:6)+Na 137 PC(16:1/16:1)+Na 138 PC(16:1/18:2)+Na 139 PC(16:1/20:4)+Na 140 PC(16:1/22:6)+K 141 PC(16:1/23:0)+Li 142 PC(16:1/24:7)+K 143 PC(16:1p/16:1)+HCOO 144 PC(16:1p/18:3)+HCOO 145 PC(16:1p/20:4)+HCOO 146 PC(16:1p/22:6)+Cl 147 PC(16:2/18:2)+K 148 PC(17:0/18:2)+Na 149 PC(17:0/20:4)+H 150 PC(17:0/20:4)+K 151 PC(17:0/20:4)+Na 152 PC(17:0/22:6)+Na 153 PC(18:0/12:0)+HCOO 154 PC(18:0/18:2)+K 155 PC(18:0/18:2)+Na 156 PC(18:0/19:2)+Na 157 PC(18:0/20:4)+CH3COO 158 PC(18:0/20:4)+K 159 PC(18:0/20:4)+Na 160 PC(18:0/22:6)+K 161 PC(18:0/22:6)+Na 162 PC(18:1/22:6)+Na

146

163 PC(18:1p/19:3)+Cl 164 PC(18:1p/21:4)+Cl 165 PC(18:1p/23:6)+Cl 166 PC(19:0/20:4)+Na 167 PC(19:1/18:2)+Na 168 PC(19:1/20:4)+Na 169 PC(20:4/20:4)+Na 170 PC(20:4/22:6)+Na 171 PC(20:4/23:6)+Cl 172 PC(22:4/22:6)+Cl 173 PC(24:5/20:4)+Cl 174 PC(30:2/18:2)+HCOO 175 PC(34:1/18:2)+Cl 176 PC(38:3/22:6)+CH3COO 177 PC(38:3/22:6)+HCOO 178 PC(38:4/22:6)+CH3COO 179 PE(15:2/23:4)+K 180 PE(16:0/18:1)-H 181 PE(16:0/18:2)+K 182 PE(16:0/18:2)+Na 183 PE(16:0/20:4)+Na 184 PE(16:0/22:6)+Na 185 PE(16:0p/20:4)-H 186 PE(16:1/20:5)-H 187 PE(17:0/20:4)-H 188 PE(17:0/22:6)-H 189 PE(17:2/23:4)+Na 190 PE(18:0/18:1)+Na 191 PE(18:0/20:4)+K 192 PE(18:0/20:4)+Na 193 PE(18:0/22:6)+K 194 PE(18:1/18:2)-H 195 PE(18:1/22:6)+Na 196 PE(18:2/17:3)-H 197 PE(18:3/18:2)-H 198 PE(19:0/20:4)-H 199 PE(20:0/19:5)+K 200 PE(20:1/22:6)-H 201 PE(37:5/20:4)-H

147

202 PEt(14:0/22:6)-H 203 PEt(15:0/22:6)-H 204 PEt(16:0/14:0)-H 205 PEt(16:0/16:2)-H 206 PEt(16:0/18:1)-H 207 PEt(16:0/18:2)-H 208 PEt(16:1/12:0)-H 209 PEt(16:1/14:0)-H 210 PEt(16:1/16:2)-H 211 PEt(16:2/16:2)-H 212 PEt(16:2/18:3)-H 213 PEt(18:0/20:4)-H 214 PEt(18:2/18:2)-H 215 PEt(18:4/16:2)-H 216 PEt(31:3/19:0)+H 217 PEt(32:2/16:2)+Na 218 PEt(4:0/18:2)-H 219 PG(15:0/18:1)-H 220 PG(16:0/16:1)-H 221 PG(16:0/18:1)-H 222 PG(16:0/18:2)-H 223 PG(16:0/22:6)-H 224 PG(16:0/23:5)+Li 225 PG(16:1/18:2)-H 226 PG(16:1/22:6)-H 227 PG(17:0/18:1)-H 228 PG(18:1/18:1)-H 229 PG(18:1/18:2)-H 230 PG(18:1/22:6)-H 231 PG(18:2/18:2)-H 232 PG(18:2/22:6)-H 233 PG(18:3/18:2)-H 234 PG(18:3/22:6)-H 235 PG(19:0/17:3)+Na 236 PG(19:0/18:3)+Li 237 PG(19:0/19:5)+Na 238 PG(19:0/20:4)+Li 239 PG(20:2/22:6)-H 240 PG(20:4/22:6)-H

148

241 PG(20:5/22:6)-H 242 PG(22:6/22:6)-H 243 PG(35:6/16:0)-H 244 PG(36:6/24:6)-H 245 PG(38:5/22:6)-H 246 phSM(d12:0/18:2)+HCOO 247 phSM(d14:0/18:2)+Cl 248 phSM(d14:1/18:2)+HCOO 249 phSM(d15:0/20:4)+Cl 250 phSM(d17:1/20:1)+Cl 251 phSM(d18:2/18:0)+Cl 252 phSM(d18:2/18:1)+Cl 253 phSM(d20:1/16:0)+Cl 254 phSM(d20:1/17:0)+Cl 255 PI(16:0/14:0)-H 256 PI(16:0/16:0)-H 257 PI(16:0/20:4)-H 258 PI(16:0/22:6)-H 259 PI(16:1/18:2)-H 260 PI(16:1/20:4)-H 261 PI(16:1p/17:0)-H 262 PI(17:0/20:4)-H 263 PI(17:0/22:6)-H 264 PI(18:0/18:2)-H 265 PI(18:0/20:1)-H 266 PI(18:0/20:4)-H 267 PI(18:0/22:6)-H 268 PI(18:0/24:6)-H 269 PI(18:0e/20:4)-H 270 PI(18:0e/22:5)-H 271 PI(18:0p/20:4)-H 272 PI(18:1/20:4)-H 273 PI(18:1/22:6)-H 274 PI(18:1p/23:2)-H 275 PI(19:0/20:4)-H 276 PI(19:0/22:6)-H 277 PI(19:1/18:0)-H 278 PI(37:1/20:3)-H 279 PI(38:1/20:3)-H

149

280 PI(38:5/22:6)+K 281 PIP(36:4/18:2)-H 282 PMe(15:0/20:4)-H 283 PMe(15:1/16:0)+(CH3CH2)3NH 284 PMe(16:0/18:1)-H 285 PMe(16:0/18:2)-H 286 PMe(16:0/20:4)-H 287 PMe(16:1/21:6)-H 288 PMe(17:1/15:0)-H 289 PMe(18:1/18:1)-H 290 PMe(24:5/20:3)-H 291 PMe(26:4/22:6)-H 292 PMe(30:0/15:0)+Na 293 PMe(30:0/17:2)+Na 294 PMe(32:2/19:0)+H 295 PS(16:0/18:2)-H 296 PS(16:0/20:4)-H 297 PS(16:0/20:5)-H 298 PS(16:0/22:6)-H 299 PS(16:0p/22:6)-H 300 PS(18:0/18:2)-H 301 PS(18:0/20:1)-H 302 PS(18:0/20:4)-H 303 PS(18:0/22:6)-H 304 PS(18:0e/20:4)-H 305 PS(18:0p/20:4)-H 306 PS(18:1/22:0)-H 307 PS(18:1/22:1)-H 308 PS(18:1/22:6)-H 309 PS(19:0/20:4)-H 310 PS(19:0/22:4)-H 311 PS(19:0/22:6)-H 312 PS(20:4/20:4)-H 313 PS(37:2/18:2)-H 314 SM(d15:0/18:1)+Cl 315 SM(d16:0/20:4)+H 316 SM(d16:0/20:4)+HCOO 317 SM(d16:0/22:4)+H 318 SM(d16:0/24:1)+Na

150

319 SM(d16:0/25:1)+Na 320 SM(d16:0/26:1)+H 321 SM(d16:0/26:2)+H 322 SM(d16:0/26:2)+Na 323 SM(d18:1/27:0)+H 324 SM(d24:0/18:1)+Na 325 TG(15:0/18:1/20:5)+H 326 TG(16:0/16:0/18:2)+Na 327 TG(16:0/18:1/18:3)+H 328 TG(16:0/18:2/20:5)+H 329 TG(16:0/20:4/22:6)+H 330 TG(16:0/20:5/20:5)+H 331 TG(16:0/20:5/22:6)+H 332 TG(17:4/18:2/18:3)+NH4 333 TG(18:1/18:2/20:4)+H 334 TG(18:1/18:2/20:5)+H 335 TG(18:1/20:4/22:6)+H 336 TG(18:1/20:5/22:6)+H 337 TG(18:2/18:2/18:2)+H 338 TG(18:3/18:2/18:3)+H 339 TG(18:3/18:2/20:4)+H 340 TG(18:4/16:0/18:2)+H 341 TG(19:3/18:2/19:4)+K

Table B.3B. Lipid IDs that were confidently detected (based on MS/MS) in shotgun and IR- MALDESI analyses of healthy rat liver. Lipid ions were filtered based on the following thresholds in LipidSearchTM: m-score threshold was set to 5 and ID quality filters were set to include lipids with grades A, B, and C. (B) lipid IDs No. only in shotgun only in IR-MALDESI both 1 CerG2(d25:0/20:4) DG(16:0/18:2) DG(18:0/20:4) 2 CerG2GNAc1(d22:1/20:1 DG(16:0/20:4) dMePE(18:0/18:2) 3 CerG3(d16:1/22:6) DG(16:1p/18:1) FA(20:4) 4 CerG3(d17:0/28:4) DG(16:1p/18:2) LPC(16:0) 5 CerG3(d24:0/18:2) DG(16:1p/20:4) LPC(16:1p) 6 ChE(20:4) DG(18:1/18:1) PC(16:0/16:1) 7 CL(10:0/20:4/22:6/18:2) DG(18:1/18:2) PC(16:0/18:2) 8 CL(16:0/18:0/18:0/18:0) DG(18:1/22:4) PC(16:0/18:3) 9 CL(17:1/18:2/18:2/18:1) DG(18:1p/18:2) PC(16:0/22:6)

151

10 CL(18:1/16:0/16:0/18:2) DG(18:1p/20:4) PC(16:1/16:1) 11 CL(18:1/16:0/18:1/16:0) dMePE(16:0/18:2) PC(16:1/22:6) 12 CL(18:1/18:1/18:2/14:0) dMePE(18:4/18:0) PC(16:1/24:7) 13 CL(18:2/14:0/18:2/18:1) FA(14:1) PC(17:0/22:6) 14 CL(18:2/15:0/18:1/18:2) LPC(18:1p) PC(18:0/18:2) 15 CL(18:2/16:0/18:0/18:0) PC(15:0/22:6) PC(18:0/20:4) 16 CL(18:2/16:0/20:4/20:4) PC(15:1/22:6) PC(18:0/22:6) 17 CL(18:2/16:1/16:1/18:2) PC(15:1/24:7) PE(16:0/18:2) 18 CL(18:2/16:1/18:2/18:2) PC(16:0/13:0) PE(16:0/20:4) 19 CL(18:2/16:1/18:2/22:6) PC(16:0/22:4) PE(16:0/22:6) 20 CL(18:2/16:1/20:4/16:1) PC(16:0/24:7) PE(18:0/18:2) 21 CL(18:2/18:0/20:4/20:3) PC(17:1/22:6) PE(18:0/20:4) 22 CL(18:2/18:1/18:1/22:6) PC(17:1/24:7) PE(18:0/22:6) 23 CL(18:2/18:1/18:2/14:0) PC(18:0/19:4) PE(18:0p/20:4) 24 CL(18:2/18:1/18:2/16:1) PC(18:1/24:7) TG(16:0/16:0/18:2) 25 CL(18:2/18:1/22:6/18:2) PC(18:1p/18:0) 26 CL(18:2/18:2/15:0/18:2) PC(18:4/16:0) 27 CL(18:2/18:2/18:2/18:2) PC(18:4/18:0) 28 CL(18:2/18:2/18:2/20:3) PC(24:5/22:6) 29 CL(18:2/18:2/20:3/18:2) Pet(16:0/20:3) 30 CL(18:2/18:2/22:1/18:1) Pet(16:1/20:4) 31 CL(18:2/20:4/18:2/18:2) Pet(18:1/20:4) 32 CL(18:2/22:0/22:4/18:2) PG(18:0/18:2) 33 CL(18:2/22:6/18:2/18:2) PG(18:0/22:6) 34 CL(18:3/16:0/20:4/18:2) PG(18:3/20:2) 35 CL(18:3/18:2/18:2/18:2) Pme(20:0/13:1) 36 CL(19:1/18:2/18:2/18:2) SM(d22:0/32:5) 37 CL(20:2/18:0/20:4/18:2) TG(13:1/21:1/21:1) 38 CL(20:4/16:0/20:1/24:1) TG(16:0/16:0/20:3) 39 CL(20:4/18:1/20:0/24:1) TG(16:0/18:1/18:2) 40 CL(21:0/16:0/16:0/18:2) TG(16:0/18:1/22:6) 41 CL(21:1/18:1/18:2/18:2) TG(16:0/18:2/18:2) 42 CL(22:1/20:4/16:0/18:2) TG(16:0/18:2/20:3) 43 CL(22:5/18:0/22:5/18:0) TG(16:0/18:2/22:6) 44 CL(22:6/16:0/18:0/22:6) TG(16:2/14:0/22:2) 45 CL(23:0/16:0/18:2/22:6) TG(16:2/16:2/24:1) 46 CL(23:0/18:0/18:0/18:0) TG(18:0/16:1/22:5) 47 CL(23:1/18:2/16:0/20:4) TG(18:1/18:1/18:2) 48 CL(23:1/18:2/20:4/18:0) TG(18:1/18:1/22:6)

152

49 CL(23:4/16:1/16:1/16:1) TG(18:1/18:2/22:6) 50 CL(24:0/16:0/18:0/16:0) TG(19:1/16:0/18:2) 51 CL(24:0/18:0/18:1/18:1) TG(19:1/17:2/19:1) 52 CL(24:0/18:0/22:5/22:5) TG(20:1/18:3/22:4) 53 CL(24:1/20:1/22:6/16:0) ZyE(0:0) 54 cPA(16:0) 55 DG(16:0p/16:0) 56 DGMG(16:1) 57 dMePE(18:0/20:4) 58 dMePE(18:0/22:6) 59 FA(20:5) 60 FA(22:6) 61 LPA(16:0) 62 LPC(12:0p) 63 LPC(18:0) 64 LPC(18:1) 65 LPC(18:2) 66 LPC(20:4) 67 LPC(22:6) 68 LPE(16:0) 69 LPE(17:1) 70 LPE(18:0) 71 LPE(20:4) 72 LPE(22:6) 73 LPG(16:0) 74 LPG(18:1) 75 LPG(18:2) 76 LPG(20:4) 77 LPG(22:6) 78 LPI(16:0) 79 LPI(18:0) 80 LPI(20:4) 81 LPI(33:0) 82 LPI(33:1) 83 LPS(18:0) 84 LPS(20:4) 85 LPS(22:6) 86 MG(20:2) 87 OAHFA(16:0/16:0)

153

88 OAHFA(16:0/17:0) 89 OAHFA(16:0/18:2) 90 OAHFA(16:0/28:3) 91 OAHFA(18:0/16:1) 92 OAHFA(18:0/17:0) 93 OAHFA(18:0/18:0) 94 OAHFA(18:1/18:0) 95 OAHFA(18:1/18:1) 96 OAHFA(18:2/18:1) 97 OAHFA(18:2/20:2) 98 OAHFA(18:2/27:1) 99 OAHFA(20:4/18:1) 100 PA(12:0/18:2) 101 PA(12:0/20:4) 102 PA(14:0/18:2) 103 PA(15:0/18:2) 104 PA(16:0p/24:2) 105 PA(18:2/22:6) 106 PA(18:2p/24:2) 107 PA(20:1p/24:2) 108 PC(10:0e/20:4) 109 PC(12:0e/18:2) 110 PC(12:0p/14:0) 111 PC(12:0p/24:7) 112 PC(14:0/20:4) 113 PC(14:0p/22:6) 114 PC(16:0/16:0) 115 PC(16:0/20:4) 116 PC(16:1/18:2) 117 PC(16:1/20:4) 118 PC(16:1/23:0) 119 PC(16:1p/16:1) 120 PC(16:1p/18:3) 121 PC(16:1p/20:4) 122 PC(16:1p/22:6) 123 PC(16:2/18:2) 124 PC(17:0/18:2) 125 PC(17:0/20:4) 126 PC(18:0/12:0)

154

127 PC(18:0/19:2) 128 PC(18:1/22:6) 129 PC(18:1p/19:3) 130 PC(18:1p/21:4) 131 PC(18:1p/23:6) 132 PC(19:0/20:4) 133 PC(19:1/18:2) 134 PC(19:1/20:4) 135 PC(20:4/20:4) 136 PC(20:4/22:6) 137 PC(20:4/23:6) 138 PC(22:4/22:6) 139 PC(24:5/20:4) 140 PC(30:2/18:2) 141 PC(34:1/18:2) 142 PC(38:3/22:6) 143 PC(38:4/22:6) 144 PE(15:2/23:4) 145 PE(16:0/18:1) 146 PE(16:0p/20:4) 147 PE(16:1/20:5) 148 PE(17:0/20:4) 149 PE(17:0/22:6) 150 PE(17:2/23:4) 151 PE(18:0/18:1) 152 PE(18:1/18:2) 153 PE(18:1/22:6) 154 PE(18:2/17:3) 155 PE(18:3/18:2) 156 PE(19:0/20:4) 157 PE(20:0/19:5) 158 PE(20:1/22:6) 159 PE(37:5/20:4) 160 Pet(14:0/22:6) 161 Pet(15:0/22:6) 162 Pet(16:0/14:0) 163 Pet(16:0/16:2) 164 Pet(16:0/18:1) 165 Pet(16:0/18:2)

155

166 Pet(16:1/12:0) 167 Pet(16:1/14:0) 168 Pet(16:1/16:2) 169 Pet(16:2/16:2) 170 Pet(16:2/18:3) 171 Pet(18:0/20:4) 172 Pet(18:2/18:2) 173 Pet(18:4/16:2) 174 Pet(31:3/19:0) 175 Pet(32:2/16:2) 176 Pet(4:0/18:2) 177 PG(15:0/18:1) 178 PG(16:0/16:1) 179 PG(16:0/18:1) 180 PG(16:0/18:2) 181 PG(16:0/22:6) 182 PG(16:0/23:5) 183 PG(16:1/18:2) 184 PG(16:1/22:6) 185 PG(17:0/18:1) 186 PG(18:1/18:1) 187 PG(18:1/18:2) 188 PG(18:1/22:6) 189 PG(18:2/18:2) 190 PG(18:2/22:6) 191 PG(18:3/18:2) 192 PG(18:3/22:6) 193 PG(19:0/17:3) 194 PG(19:0/18:3) 195 PG(19:0/19:5) 196 PG(19:0/20:4) 197 PG(20:2/22:6) 198 PG(20:4/22:6) 199 PG(20:5/22:6) 200 PG(22:6/22:6) 201 PG(35:6/16:0) 202 PG(36:6/24:6) 203 PG(38:5/22:6) 204 phSM(d12:0/18:2)

156

205 phSM(d14:0/18:2) 206 phSM(d14:1/18:2) 207 phSM(d15:0/20:4) 208 phSM(d17:1/20:1) 209 phSM(d18:2/18:0) 210 phSM(d18:2/18:1) 211 phSM(d20:1/16:0) 212 phSM(d20:1/17:0) 213 PI(16:0/14:0) 214 PI(16:0/16:0) 215 PI(16:0/20:4) 216 PI(16:0/22:6) 217 PI(16:1/18:2) 218 PI(16:1/20:4) 219 PI(16:1p/17:0) 220 PI(17:0/20:4) 221 PI(17:0/22:6) 222 PI(18:0/18:2) 223 PI(18:0/20:1) 224 PI(18:0/20:4) 225 PI(18:0/22:6) 226 PI(18:0/24:6) 227 PI(18:0e/20:4) 228 PI(18:0e/22:5) 229 PI(18:0p/20:4) 230 PI(18:1/20:4) 231 PI(18:1/22:6) 232 PI(18:1p/23:2) 233 PI(19:0/20:4) 234 PI(19:0/22:6) 235 PI(19:1/18:0) 236 PI(37:1/20:3) 237 PI(38:1/20:3) 238 PI(38:5/22:6) 239 PIP(36:4/18:2) 240 Pme(15:0/20:4) 241 Pme(15:1/16:0) 242 Pme(16:0/18:1) 243 Pme(16:0/18:2)

157

244 Pme(16:0/20:4) 245 Pme(16:1/21:6) 246 Pme(17:1/15:0) 247 Pme(18:1/18:1) 248 Pme(24:5/20:3) 249 Pme(26:4/22:6) 250 Pme(30:0/15:0) 251 Pme(30:0/17:2) 252 Pme(32:2/19:0) 253 PS(16:0/18:2) 254 PS(16:0/20:4) 255 PS(16:0/20:5) 256 PS(16:0/22:6) 257 PS(16:0p/22:6) 258 PS(18:0/18:2) 259 PS(18:0/20:1) 260 PS(18:0/20:4) 261 PS(18:0/22:6) 262 PS(18:0e/20:4) 263 PS(18:0p/20:4) 264 PS(18:1/22:0) 265 PS(18:1/22:1) 266 PS(18:1/22:6) 267 PS(19:0/20:4) 268 PS(19:0/22:4) 269 PS(19:0/22:6) 270 PS(20:4/20:4) 271 PS(37:2/18:2) 272 SM(d15:0/18:1) 273 SM(d16:0/20:4) 274 SM(d16:0/22:4) 275 SM(d16:0/24:1) 276 SM(d16:0/25:1) 277 SM(d16:0/26:1) 278 SM(d16:0/26:2) 279 SM(d18:1/27:0) 280 SM(d24:0/18:1) 281 TG(15:0/18:1/20:5) 282 TG(16:0/18:1/18:3)

158

283 TG(16:0/18:2/20:5) 284 TG(16:0/20:4/22:6) 285 TG(16:0/20:5/20:5) 286 TG(16:0/20:5/22:6) 287 TG(17:4/18:2/18:3) 288 TG(18:1/18:2/20:4) 289 TG(18:1/18:2/20:5) 290 TG(18:1/20:4/22:6) 291 TG(18:1/20:5/22:6) 292 TG(18:2/18:2/18:2) 293 TG(18:3/18:2/18:3) 294 TG(18:3/18:2/20:4) 295 TG(18:4/16:0/18:2) 296 TG(19:3/18:2/19:4)

Table B.3C. Lipid classes that were confidently detected (based on MS/MS) in shotgun and IR- MALDESI analyses of healthy rat liver. Lipid ions were filtered based on the following thresholds in LipidSearchTM: m-score threshold was set to 5 and ID quality filters were set to include lipids with grades A, B, and C. I lipid classes No. only in shotgun only in IR-MALDESI both 1 CerG2 ZyE DG 2 CerG2GNAc1 dMePE 3 CerG3 FA 4 ChE LPC 5 CL PC 6 cPA PE 7 DGMG PEt 8 LPA PG 9 LPE PMe 10 LPG SM 11 LPI TG 12 LPS 13 MG 14 OAHFA 15 PA 16 phSM 17 PI 18 PIP 19 PS

159

Table B.4A. Lipid IDs, from IR-MALDESI analyses, putatively (based on MS1 only) annotated using METASPACE software. IR-MALDESI formula adduct molecular name C31H54NO10P M+Na PS(22:3(10Z,13Z,16Z)/3:0), PS(3:0/22:3(10Z,13Z,16Z)) PG(3:0/22:5(4Z,7Z,10Z,13Z,16Z)), Lysobisphosphatidate (3:0/0:0/22:5(4Z,7Z,10Z,13Z,16Z)/0:0), PG(22:5(7Z,10Z,13Z,16Z,19Z)/3:0), C31H51O10P M+Na PG(22:5(4Z,7Z,10Z,13Z,16Z)/3:0), Lysobisphosphatidate (3:0/0:0/22:5(7Z,10Z,13Z,16Z,19Z)/0:0), PG(3:0/22:5(7Z,10Z,13Z,16Z,19Z)) PA(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), PA(18:1(6Z)/22:4(7Z,10Z,13Z,16Z)), PA(16:1(9Z)/24:4(9Z,12Z,15Z,18Z)), PA(22:3(10Z,13Z,16Z)/18:2(9Z,12Z)), PA(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), PA(18:0/22:5(7Z,10Z,13Z,16Z,19Z)), PA(26:4(11Z,14Z,17Z,20Z)/14:1(9Z)), PA(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PA(22:5(7Z,10Z,13Z,16Z,19Z)/18:0), PA(22:4(7Z,10Z,13Z,16Z)/18:1(11E)), PA(20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), PA(14:1(9Z)/26:4(11Z,14Z,17Z,20Z)), PA(12:0/28:5(10Z,13Z,16Z,19Z,22Z)), PA(16:0/24:5(9Z,12Z,15Z,18Z,21Z)), PA(2:0/38:5(20Z,23Z,26Z,29Z,32Z)), PA(18:1(11E)/22:4(7Z,10Z,13Z,16Z)), C43H75O8P M+K PA(22:4(7Z,10Z,13Z,16Z)/18:1(6Z)), PA(10:0/30:5(15Z,18Z,21Z,24Z,27Z)), PA(18:0/22:5(4Z,7Z,10Z,13Z,16Z)), PA(8:0/32:5(14Z,17Z,20Z,23Z,26Z)), PA(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), PA(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PA(18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), PA(4:0/36:5(21Z,24Z,27Z,30Z,33Z)), PA(12:0/28:5(13Z,16Z,19Z,22Z,25Z)), PA(6:0/34:5(16Z,19Z,22Z,25Z,28Z)), PA(32:5(14Z,17Z,20Z,23Z,26Z)/8:0), PA(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PA(30:5(12Z,15Z,18Z,21Z,24Z)/10:0), PA(22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), PA(14:0/26:5(8Z,11Z,14Z,17Z,20Z)), PA(22:5(4Z,7Z,10Z,13Z,16Z)/18:0), PA(36:5(18Z,21Z,24Z,27Z,30Z)/4:0),

160

PA(22:3(10Z,13Z,16Z)/18:2(9Z,11E)), PA(16:1(6Z)/24:4(9Z,12Z,15Z,18Z)), PA(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), PA(8:0/32:5(17Z,20Z,23Z,26Z,29Z)), PA(20:3(11Z,14Z,17Z)/20:2(11Z,14Z)), PA(28:5(13Z,16Z,19Z,22Z,25Z)/12:0), PA(30:5(15Z,18Z,21Z,24Z,27Z)/10:0), PA(34:5(19Z,22Z,25Z,28Z,31Z)/6:0), PA(20:3(8Z,11Z,14Z)/20:2(11Z,14Z)), PA(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PA(20:2(11Z,14Z)/20:3(11Z,14Z,17Z)), PA(2:0/38:5(23Z,26Z,29Z,32Z,35Z)), PA(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PA(34:5(16Z,19Z,22Z,25Z,28Z)/6:0), PA(28:5(10Z,13Z,16Z,19Z,22Z)/12:0), PA(26:5(11Z,14Z,17Z,20Z,23Z)/14:0), PA(32:5(17Z,20Z,23Z,26Z,29Z)/8:0)

161

PC(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PC(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), PC(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:3(5Z,8Z,11Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/20:4(8Z,11Z,14Z,17Z)), PC(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:3(8Z,11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), PC(20:4(5Z,8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PC(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PC(20:4(8Z,11Z,14Z,17Z)/20:3(5Z,8Z,11Z)), PC(20:4(8Z,11Z,14Z,17Z)/20:3(8Z,11Z,14Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PC(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), C48H82NO8P M+H PC(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe(20:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:2(11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(20:2(11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:3(5Z,8Z,11Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(20:3(8Z,11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/22:2(13Z,16Z)), Pe- NMe(22:2(13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/20:3(5Z,8Z,11Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/20:3(8Z,11Z,14Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/20:2(11Z,14Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:1(11Z))

162

PE(20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(20:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(20:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(20:2(11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), PE(20:4(5Z,8Z,11Z,14Z)/22:2(13Z,16Z)), PE(20:4(8Z,11Z,14Z,17Z)/22:2(13Z,16Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/22:1(13Z)), PE(22:1(13Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(22:2(13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PE(22:2(13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:4(7Z,10Z,13Z,16Z)/20:2(11Z,14Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/20:1(11Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/20:1(11Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:0), Pe- NMe2(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- C47H82NO8P M+H NMe2(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), Pe- NMe2(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), Pe- NMe2(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0)

163

PC(16:0/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/20:3(5Z,8Z,11Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:1(11Z)), PC(18:3(9Z,12Z,15Z)/20:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PC(20:1(11Z)/18:3(6Z,9Z,12Z)), PC(20:1(11Z)/18:3(9Z,12Z,15Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/18:1(9Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(20:4(8Z,11Z,14Z,17Z)/18:0), PC(22:4(7Z,10Z,13Z,16Z)/16:0), Pe-NMe(18:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:2(9Z,12Z)/22:2(13Z,16Z)), Pe- C46H84NO8P M+H NMe(18:3(6Z,9Z,12Z)/22:1(13Z)), Pe- NMe(18:3(9Z,12Z,15Z)/22:1(13Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:0), Pe- NMe(20:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:1(11Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:0), Pe- NMe(22:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:1(13Z)/18:3(6Z,9Z,12Z)), Pe- NMe(22:1(13Z)/18:3(9Z,12Z,15Z)), Pe- NMe(22:2(13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:0)

164

PC(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), PC(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), PC(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), PC(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:2(11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:2(11Z,14Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:1(11Z)), PC(20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:2(11Z,14Z)/18:3(6Z,9Z,12Z)), PC(20:2(11Z,14Z)/18:3(9Z,12Z,15Z)), PC(20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), PC(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:1(11Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:1(9Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:0), C46H82NO8P M+H PC(22:4(7Z,10Z,13Z,16Z)/16:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/16:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(18:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), Pe- NMe(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), Pe- NMe(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:1(11Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:2(11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:2(11Z,14Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), Pe- NMe(22:1(13Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), Pe- NMe(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), Pe-

165

NMe(22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:0), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:0)

166

PC(15:0/22:4(7Z,10Z,13Z,16Z)), PC(22:4(7Z,10Z,13Z,16Z)/15:0), PE(18:0/22:4(7Z,10Z,13Z,16Z)), PE(18:2(9Z,12Z)/22:2(13Z,16Z)), PE(18:3(6Z,9Z,12Z)/22:1(13Z)), PE(18:3(9Z,12Z,15Z)/22:1(13Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:0), PE(20:0/20:4(5Z,8Z,11Z,14Z)), PE(20:0/20:4(8Z,11Z,14Z,17Z)), PE(20:1(11Z)/20:3(5Z,8Z,11Z)), PE(20:1(11Z)/20:3(8Z,11Z,14Z)), PE(20:2(11Z,14Z)/20:2(11Z,14Z)), PE(20:3(5Z,8Z,11Z)/20:1(11Z)), PE(20:3(8Z,11Z,14Z)/20:1(11Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:0), PE(20:4(8Z,11Z,14Z,17Z)/20:0), PE(22:0/18:4(6Z,9Z,12Z,15Z)), PE(22:1(13Z)/18:3(6Z,9Z,12Z)), PE(22:1(13Z)/18:3(9Z,12Z,15Z)), PE(22:2(13Z,16Z)/18:2(9Z,12Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:0), Pe- NMe2(16:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(18:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:0/20:4(8Z,11Z,14Z,17Z)), Pe- C45H82NO8P M+H NMe2(18:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:2(9Z,12Z)/20:2(11Z,14Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:1(11Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:1(11Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:0), Pe- NMe2(20:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(20:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:2(11Z,14Z)/18:2(9Z,12Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:1(11Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:1(11Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/16:0)

167

PC(15:0/22:5(4Z,7Z,10Z,13Z,16Z)), PC(15:0/22:5(7Z,10Z,13Z,16Z,19Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/15:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/15:0), PE(18:0/22:5(4Z,7Z,10Z,13Z,16Z)), PE(18:0/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PE(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), PE(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:1(11Z)/20:4(8Z,11Z,14Z,17Z)), PE(20:2(11Z,14Z)/20:3(5Z,8Z,11Z)), PE(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PE(20:3(5Z,8Z,11Z)/20:2(11Z,14Z)), PE(20:3(8Z,11Z,14Z)/20:2(11Z,14Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PE(22:1(13Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), C45H80NO8P M+H PE(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:0), PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:0), Pe- NMe2(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(18:0/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:2(11Z,14Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:2(11Z,14Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:1(11Z)), Pe- NMe2(20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(20:2(11Z,14Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:2(11Z,14Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), Pe-

168

NMe2(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:1(11Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:1(9Z))

169

PC(15:0/20:2(11Z,14Z)), PC(20:2(11Z,14Z)/15:0), PE(14:1(9Z)/24:1(15Z)), PE(16:0/22:2(13Z,16Z)), PE(16:1(9Z)/22:1(13Z)), PE(18:0/20:2(11Z,14Z)), PE(18:1(11Z)/20:1(11Z)), PE(18:1(9Z)/20:1(11Z)), PE(18:2(9Z,12Z)/20:0), PE(20:0/18:2(9Z,12Z)), PE(20:1(11Z)/18:1(11Z)), PE(20:1(11Z)/18:1(9Z)), PE(20:2(11Z,14Z)/18:0), PE(22:1(13Z)/16:1(9Z)), PE(22:2(13Z,16Z)/16:0), PE(24:1(15Z)/14:1(9Z)), Pe- C43H82NO8P M+Na NMe2(18:1(9Z)/18:1(9Z)), Pe-NMe(15:0/22:2(13Z,16Z)), Pe- NMe(22:2(13Z,16Z)/15:0), Pe-NMe2(16:1(9Z)/20:1(11Z)), Pe- NMe2(18:1(9Z)/18:1(11Z)), Pe-NMe2(18:1(11Z)/18:1(9Z)), Pe- NMe2(18:1(11Z)/18:1(11Z)), Pe-NMe2(14:0/22:2(13Z,16Z)), Pe- NMe2(14:1(9Z)/22:1(13Z)), Pe-NMe2(16:0/20:2(11Z,14Z)), Pe- NMe2(18:0/18:2(9Z,12Z)), Pe-NMe2(18:2(9Z,12Z)/18:0), Pe- NMe2(20:1(11Z)/16:1(9Z)), Pe-NMe2(20:2(11Z,14Z)/16:0), Pe- NMe2(22:1(13Z)/14:1(9Z)), Pe-NMe2(22:2(13Z,16Z)/14:0)

170

PA(18:0/22:5(4Z,7Z,10Z,13Z,16Z)), PA(18:0/22:5(7Z,10Z,13Z,16Z,19Z)), PA(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), PA(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PA(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PA(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), PA(18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), PA(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), PA(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PA(20:1(11Z)/20:4(8Z,11Z,14Z,17Z)), PA(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), PA(20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), C43H75O8P M+K PA(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PA(22:1(13Z)/18:4(6Z,9Z,12Z,15Z)), PA(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), PA(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), PA(22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), PA(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), PA(22:5(4Z,7Z,10Z,13Z,16Z)/18:0), PA(22:5(7Z,10Z,13Z,16Z,19Z)/18:0), PA(20:2(11Z,14Z)/20:3(5Z,8Z,11Z)), PA(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PA(20:3(5Z,8Z,11Z)/20:2(11Z,14Z)), PA(20:3(8Z,11Z,14Z)/20:2(11Z,14Z))

171

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine, Pe-NMe(14:1(9Z)/24:1(15Z)), Pe- C44H84NO8P M+H NMe(16:0/22:2(13Z,16Z)), Pe-NMe(16:1(9Z)/22:1(13Z)), Pe- NMe(18:0/20:2(11Z,14Z)), Pe-NMe(18:1(11Z)/20:1(11Z)), Pe- NMe(18:1(9Z)/20:1(11Z)), Pe-NMe(18:2(9Z,12Z)/20:0), Pe- NMe(20:0/18:2(9Z,12Z)), Pe-NMe(20:1(11Z)/18:1(11Z)), Pe- NMe(20:1(11Z)/18:1(9Z)), Pe-NMe(20:2(11Z,14Z)/18:0), Pe- NMe(22:1(13Z)/16:1(9Z)), Pe-NMe(22:2(13Z,16Z)/16:0), Pe- NMe(24:1(15Z)/14:1(9Z)), Pe-NMe2(15:0/22:2(13Z,16Z)), Pe- NMe2(22:2(13Z,16Z)/15:0)

172

PC(14:1(9Z)/22:2(13Z,16Z)), PC(16:0/20:3(5Z,8Z,11Z)), PC(16:0/20:3(8Z,11Z,14Z)), PC(16:1(9Z)/20:2(11Z,14Z)), PC(18:0/18:3(6Z,9Z,12Z)), PC(18:0/18:3(9Z,12Z,15Z)), PC(18:1(11Z)/18:2(9Z,12Z)), PC(18:1(9Z)/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:1(9Z)), PC(18:3(6Z,9Z,12Z)/18:0), PC(18:3(9Z,12Z,15Z)/18:0), PC(20:2(11Z,14Z)/16:1(9Z)), PC(20:3(5Z,8Z,11Z)/16:0), PC(20:3(8Z,11Z,14Z)/16:0), PC(22:2(13Z,16Z)/14:1(9Z)), Pe- NMe(16:1(9Z)/22:2(13Z,16Z)), Pe-NMe(18:0/20:3(5Z,8Z,11Z)), Pe-NMe(18:0/20:3(8Z,11Z,14Z)), Pe- C44H82NO8P M+H NMe(18:1(11Z)/20:2(11Z,14Z)), Pe- NMe(18:1(9Z)/20:2(11Z,14Z)), Pe- NMe(18:2(9Z,12Z)/20:1(11Z)), Pe-NMe(18:3(6Z,9Z,12Z)/20:0), Pe-NMe(18:3(9Z,12Z,15Z)/20:0), Pe- NMe(20:0/18:3(6Z,9Z,12Z)), Pe-NMe(20:0/18:3(9Z,12Z,15Z)), Pe-NMe(20:1(11Z)/18:2(9Z,12Z)), Pe- NMe(20:2(11Z,14Z)/18:1(11Z)), Pe- NMe(20:2(11Z,14Z)/18:1(9Z)), Pe-NMe(20:3(5Z,8Z,11Z)/18:0), Pe-NMe(20:3(8Z,11Z,14Z)/18:0), Pe- NMe(22:2(13Z,16Z)/16:1(9Z))

173

PC(14:0/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(16:0/20:4(8Z,11Z,14Z,17Z)), PC(16:1(9Z)/20:3(5Z,8Z,11Z)), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PC(18:1(11Z)/18:3(6Z,9Z,12Z)), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(18:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(20:3(5Z,8Z,11Z)/16:1(9Z)), PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PC(20:4(8Z,11Z,14Z,17Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe(16:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(18:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(18:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(18:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:1(9Z)/20:3(5Z,8Z,11Z)), Pe- C44H80NO8P M+H NMe(18:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:2(9Z,12Z)/20:2(11Z,14Z)), Pe- NMe(18:3(6Z,9Z,12Z)/20:1(11Z)), Pe- NMe(18:3(9Z,12Z,15Z)/20:1(11Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/20:0), Pe- NMe(20:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(20:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe(20:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe(20:2(11Z,14Z)/18:2(9Z,12Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:1(11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:1(9Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:1(9Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/18:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/18:0), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe2(15:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/15:0)

174

PC(15:0/20:3(5Z,8Z,11Z)), PC(15:0/20:3(8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/15:0), PC(20:3(8Z,11Z,14Z)/15:0), PE(16:1(9Z)/22:2(13Z,16Z)), PE(18:0/20:3(5Z,8Z,11Z)), PE(18:0/20:3(8Z,11Z,14Z)), PE(18:1(11Z)/20:2(11Z,14Z)), PE(18:1(9Z)/20:2(11Z,14Z)), PE(18:2(9Z,12Z)/20:1(11Z)), PE(18:3(6Z,9Z,12Z)/20:0), PE(18:3(9Z,12Z,15Z)/20:0), PE(20:0/18:3(6Z,9Z,12Z)), PE(20:0/18:3(9Z,12Z,15Z)), PE(20:1(11Z)/18:2(9Z,12Z)), PE(20:2(11Z,14Z)/18:1(11Z)), PE(20:2(11Z,14Z)/18:1(9Z)), PE(20:3(5Z,8Z,11Z)/18:0), PE(20:3(8Z,11Z,14Z)/18:0), PE(22:2(13Z,16Z)/16:1(9Z)), Pe- NMe2(14:1(9Z)/22:2(13Z,16Z)), Pe- C43H80NO8P M+H NMe2(16:0/20:3(5Z,8Z,11Z)), Pe-NMe2(16:0/20:3(8Z,11Z,14Z)), Pe-NMe2(16:1(9Z)/20:2(11Z,14Z)), Pe- NMe2(18:0/18:3(6Z,9Z,12Z)), Pe-NMe2(18:0/18:3(9Z,12Z,15Z)), Pe-NMe2(18:1(11Z)/18:2(9Z,12Z)), Pe- NMe2(18:1(9Z)/18:2(9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/18:1(11Z)), Pe- NMe2(18:2(9Z,12Z)/18:1(9Z)), Pe-NMe2(18:3(6Z,9Z,12Z)/18:0), Pe-NMe2(18:3(9Z,12Z,15Z)/18:0), Pe- NMe2(20:2(11Z,14Z)/16:1(9Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/16:0), Pe-NMe2(20:3(8Z,11Z,14Z)/16:0), Pe-NMe2(22:2(13Z,16Z)/14:1(9Z))

175

PC(15:0/20:4(5Z,8Z,11Z,14Z)), PC(15:0/20:4(8Z,11Z,14Z,17Z)), PC(20:4(5Z,8Z,11Z,14Z)/15:0), PC(20:4(8Z,11Z,14Z,17Z)/15:0), PE(16:0/22:4(7Z,10Z,13Z,16Z)), PE(16:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:0/20:4(5Z,8Z,11Z,14Z)), PE(18:0/20:4(8Z,11Z,14Z,17Z)), PE(18:1(11Z)/20:3(5Z,8Z,11Z)), PE(18:1(11Z)/20:3(8Z,11Z,14Z)), PE(18:1(9Z)/20:3(5Z,8Z,11Z)), PE(18:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:2(9Z,12Z)/20:2(11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:1(11Z)), PE(18:3(9Z,12Z,15Z)/20:1(11Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:0), PE(20:0/18:4(6Z,9Z,12Z,15Z)), PE(20:1(11Z)/18:3(6Z,9Z,12Z)), PE(20:1(11Z)/18:3(9Z,12Z,15Z)), PE(20:2(11Z,14Z)/18:2(9Z,12Z)), PE(20:3(5Z,8Z,11Z)/18:1(11Z)), PE(20:3(5Z,8Z,11Z)/18:1(9Z)), PE(20:3(8Z,11Z,14Z)/18:1(11Z)), PE(20:3(8Z,11Z,14Z)/18:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:0), PE(20:4(8Z,11Z,14Z,17Z)/18:0), PE(22:4(7Z,10Z,13Z,16Z)/16:0), Pe-NMe(15:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/15:0), Pe- NMe2(14:0/22:4(7Z,10Z,13Z,16Z)), Pe- C43H78NO8P M+H NMe2(16:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(16:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(16:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:2(9Z,12Z)/18:2(9Z,12Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:1(11Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:1(9Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:1(11Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:1(9Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:0), Pe- NMe2(20:3(5Z,8Z,11Z)/16:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/16:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/16:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/16:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe2(16:0/20:4(8Z,11Z,14Z,17Z))

176

PC(15:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/15:0), PE(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), PE(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), PE(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:0/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), PE(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:2(11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:2(11Z,14Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:1(11Z)), PE(20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:2(11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:2(11Z,14Z)/18:3(9Z,12Z,15Z)), PE(20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), PE(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:1(11Z)), C43H76NO8P M+H PE(20:4(8Z,11Z,14Z,17Z)/18:1(9Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:0), PE(22:4(7Z,10Z,13Z,16Z)/16:1(9Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/16:0), PE(22:5(7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(15:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(15:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/15:0), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/15:0), Pe- NMe2(14:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(14:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(14:1(9Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(16:0/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(16:1(9Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(16:1(9Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:1(11Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:1(9Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:2(9Z,12Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:2(9Z,12Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:2(9Z,12Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:1(11Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:1(9Z)), Pe-

177

NMe2(20:4(5Z,8Z,11Z,14Z)/16:1(9Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/16:1(9Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/16:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/14:1(9Z))

178

PE(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:2(9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:3(6Z,9Z,12Z)/20:3(5Z,8Z,11Z)), PE(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PE(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:3(5Z,8Z,11Z)/18:3(6Z,9Z,12Z)), PE(20:3(5Z,8Z,11Z)/18:3(9Z,12Z,15Z)), PE(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(11Z)), C43H74NO8P M+H PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/16:1(9Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/15:0), Pe- NMe2(14:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(14:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(14:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(16:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(18:2(9Z,12Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:2(9Z,12Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/16:1(9Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/14:1(9Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/14:1(9Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/14:0)

179

PC(15:0/18:3(6Z,9Z,12Z)), PC(15:0/18:3(9Z,12Z,15Z)), PC(18:3(6Z,9Z,12Z)/15:0), PC(18:3(9Z,12Z,15Z)/15:0), PE(14:1(9Z)/22:2(13Z,16Z)), PE(16:0/20:3(5Z,8Z,11Z)), PE(16:0/20:3(8Z,11Z,14Z)), PE(16:1(9Z)/20:2(11Z,14Z)), PE(18:0/18:3(6Z,9Z,12Z)), PE(18:0/18:3(9Z,12Z,15Z)), PE(18:1(11Z)/18:2(9Z,12Z)), PE(18:1(9Z)/18:2(9Z,12Z)), PE(18:2(9Z,12Z)/18:1(11Z)), PE(18:2(9Z,12Z)/18:1(9Z)), PE(18:3(6Z,9Z,12Z)/18:0), PE(18:3(9Z,12Z,15Z)/18:0), PE(20:2(11Z,14Z)/16:1(9Z)), PE(20:3(5Z,8Z,11Z)/16:0), PE(20:3(8Z,11Z,14Z)/16:0), PE(22:2(13Z,16Z)/14:1(9Z)), Pe- C41H76NO8P M+Na NMe(15:0/20:3(5Z,8Z,11Z)), Pe-NMe(15:0/20:3(8Z,11Z,14Z)), Pe-NMe(20:3(5Z,8Z,11Z)/15:0), Pe- NMe(20:3(8Z,11Z,14Z)/15:0), Pe-NMe2(14:0/20:3(5Z,8Z,11Z)), Pe-NMe2(14:0/20:3(8Z,11Z,14Z)), Pe- NMe2(14:1(9Z)/20:2(11Z,14Z)), Pe- NMe2(16:0/18:3(6Z,9Z,12Z)), Pe-NMe2(16:0/18:3(9Z,12Z,15Z)), Pe-NMe2(16:1(9Z)/18:2(9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/16:1(9Z)), Pe-NMe2(18:3(6Z,9Z,12Z)/16:0), Pe-NMe2(18:3(9Z,12Z,15Z)/16:0), Pe- NMe2(20:2(11Z,14Z)/14:1(9Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/14:0), Pe-NMe2(20:3(8Z,11Z,14Z)/14:0)

180

PC(14:0/20:2(11Z,14Z)), PC(14:1(9Z)/20:1(11Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(16:1(9Z)/18:1(9Z)), PC(18:1(11Z)/16:1(9Z)), PC(18:1(9Z)/16:1(9Z)), PC(18:2(9Z,12Z)/16:0), PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PE(15:0/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/15:0), Pe- NMe(18:1(9Z)/18:1(9Z)), Pe-NMe(16:1(9Z)/20:1(11Z)), Pe- C42H80NO8P M+H NMe(18:1(9Z)/18:1(11Z)), Pe-NMe(18:1(11Z)/18:1(9Z)), Pe- NMe(18:1(11Z)/18:1(11Z)), Pe-NMe(14:0/22:2(13Z,16Z)), Pe- NMe(14:1(9Z)/22:1(13Z)), Pe-NMe(16:0/20:2(11Z,14Z)), Pe- NMe(18:0/18:2(9Z,12Z)), Pe-NMe(18:2(9Z,12Z)/18:0), Pe- NMe(20:1(11Z)/16:1(9Z)), Pe-NMe(20:2(11Z,14Z)/16:0), Pe- NMe(22:1(13Z)/14:1(9Z)), Pe-NMe(22:2(13Z,16Z)/14:0), Pe- NMe2(15:0/20:2(11Z,14Z)), Pe-NMe2(20:2(11Z,14Z)/15:0)

PC(15:0/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/15:0), PE(14:0/22:2(13Z,16Z)), PE(14:1(9Z)/22:1(13Z)), PE(16:0/20:2(11Z,14Z)), PE(16:1(9Z)/20:1(11Z)), PE(18:0/18:2(9Z,12Z)), PE(18:1(11Z)/18:1(11Z)), PE(18:1(11Z)/18:1(9Z)), PE(18:1(9Z)/18:1(11Z)), PE(18:1(9Z)/18:1(9Z)), PE(18:2(9Z,12Z)/18:0), PE(20:1(11Z)/16:1(9Z)), PE(20:2(11Z,14Z)/16:0), C41H78NO8P M+H PE(22:1(13Z)/14:1(9Z)), PE(22:2(13Z,16Z)/14:0), Pe- NMe(15:0/20:2(11Z,14Z)), Pe-NMe(20:2(11Z,14Z)/15:0), Pe- NMe2(14:1(9Z)/20:1(11Z)), Pe-NMe2(16:1(9Z)/18:1(9Z)), Pe- NMe2(16:1(9Z)/18:1(11Z)), Pe-NMe2(14:0/20:2(11Z,14Z)), Pe- NMe2(16:0/18:2(9Z,12Z)), Pe-NMe2(18:1(9Z)/16:1(9Z)), Pe- NMe2(18:1(11Z)/16:1(9Z)), Pe-NMe2(18:2(9Z,12Z)/16:0), Pe- NMe2(20:1(11Z)/14:1(9Z)), Pe-NMe2(20:2(11Z,14Z)/14:0)

181

PC(15:0/18:4(6Z,9Z,12Z,15Z)), PC(18:4(6Z,9Z,12Z,15Z)/15:0), PE(14:0/22:4(7Z,10Z,13Z,16Z)), PE(16:0/20:4(5Z,8Z,11Z,14Z)), PE(16:0/20:4(8Z,11Z,14Z,17Z)), PE(16:1(9Z)/20:3(5Z,8Z,11Z)), PE(18:0/18:4(6Z,9Z,12Z,15Z)), PE(18:1(11Z)/18:3(6Z,9Z,12Z)), PE(18:1(11Z)/18:3(9Z,12Z,15Z)), PE(18:1(9Z)/18:3(6Z,9Z,12Z)), PE(18:1(9Z)/18:3(9Z,12Z,15Z)), PE(18:2(9Z,12Z)/18:2(9Z,12Z)), PE(18:3(6Z,9Z,12Z)/18:1(11Z)), PE(18:3(6Z,9Z,12Z)/18:1(9Z)), PE(18:3(9Z,12Z,15Z)/18:1(11Z)), PE(18:3(9Z,12Z,15Z)/18:1(9Z)), PE(18:4(6Z,9Z,12Z,15Z)/18:0), PE(20:3(5Z,8Z,11Z)/16:1(9Z)), PE(20:3(8Z,11Z,14Z)/16:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/16:0), PE(20:4(8Z,11Z,14Z,17Z)/16:0), PE(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe(15:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(15:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/15:0), Pe- C41H74NO8P M+H NMe(20:4(8Z,11Z,14Z,17Z)/15:0), Pe- NMe2(14:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(14:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(14:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(14:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(16:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(16:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(16:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/16:1(9Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/16:1(9Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/16:0), Pe- NMe2(20:3(5Z,8Z,11Z)/14:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/14:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/14:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/14:0)

PC(14:0/18:2(9Z,12Z)), PC(14:1(9Z)/18:1(11Z)), PC(14:1(9Z)/18:1(9Z)), PC(16:1(9Z)/16:1(9Z)), PC(18:1(11Z)/14:1(9Z)), PC(18:1(9Z)/14:1(9Z)), PC(18:2(9Z,12Z)/14:0), PE(15:0/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/15:0), Pe-NMe(14:1(9Z)/20:1(11Z)), Pe- C40H76NO8P M+H NMe(16:1(9Z)/18:1(9Z)), Pe-NMe(16:1(9Z)/18:1(11Z)), Pe- NMe(14:0/20:2(11Z,14Z)), Pe-NMe(16:0/18:2(9Z,12Z)), Pe- NMe(18:1(9Z)/16:1(9Z)), Pe-NMe(18:1(11Z)/16:1(9Z)), Pe- NMe(18:2(9Z,12Z)/16:0), Pe-NMe(20:1(11Z)/14:1(9Z)), Pe- NMe(20:2(11Z,14Z)/14:0), Pe-NMe2(15:0/18:2(9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/15:0)

182

PE(14:0/20:2(11Z,14Z)), PE(14:1(9Z)/20:1(11Z)), PE(16:0/18:2(9Z,12Z)), PE(16:1(9Z)/18:1(11Z)), PE(16:1(9Z)/18:1(9Z)), PE(18:1(11Z)/16:1(9Z)), PE(18:1(9Z)/16:1(9Z)), PE(18:2(9Z,12Z)/16:0), PE(20:1(11Z)/14:1(9Z)), PE(20:2(11Z,14Z)/14:0), Pe- C39H74NO8P M+H NMe(15:0/18:2(9Z,12Z)), Pe-NMe(18:2(9Z,12Z)/15:0), Pe- NMe2(14:1(9Z)/18:1(9Z)), Pe-NMe2(14:1(9Z)/18:1(11Z)), Pe- NMe2(16:1(9Z)/16:1(9Z)), Pe-NMe2(14:0/18:2(9Z,12Z)), Pe- NMe2(18:1(11Z)/14:1(9Z)), Pe-NMe2(18:1(9Z)/14:1(9Z)), Pe- NMe2(18:2(9Z,12Z)/14:0)

C40H49N5O6 M+H Jubanine A C23H46N6O13 M+Na Neomycin C36H60O3 M+H Momordicilin

183

PE(18:1(11Z)/22:2(13Z,16Z)), PE(18:1(9Z)/22:2(13Z,16Z)), PE(18:2(9Z,12Z)/22:1(13Z)), PE(18:3(6Z,9Z,12Z)/22:0), PE(18:3(9Z,12Z,15Z)/22:0), PE(20:0/20:3(5Z,8Z,11Z)), PE(20:0/20:3(8Z,11Z,14Z)), PE(20:1(11Z)/20:2(11Z,14Z)), C45H84NO8P M+Na PE(20:2(11Z,14Z)/20:1(11Z)), PE(20:3(5Z,8Z,11Z)/20:0), PE(20:3(8Z,11Z,14Z)/20:0), PE(22:0/18:3(6Z,9Z,12Z)), PE(22:0/18:3(9Z,12Z,15Z)), PE(22:1(13Z)/18:2(9Z,12Z)), PE(22:2(13Z,16Z)/18:1(11Z)), PE(22:2(13Z,16Z)/18:1(9Z))

184

185

186

187

188

PA(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PA(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PA(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), PA(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), PA(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PA(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), C43H75O8P M+K PA(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PA(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), PA(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), PA(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), PA(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PA(20:3(8Z,11Z,14Z)/20:2(11Z,14Z))

Ginsenoside Rg5, (20E)-Ginsenoside F4, Hebevinoside X, C42H70O12 M+Na Ginsenoside Rg6 PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), C44H84NO8P M+H PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine

PC(14:0/20:0), PC(16:0/18:0), PC(18:0/16:0), PC(20:0/14:0), C42H84NO8P M+Na PE(15:0/22:0), PE(22:0/15:0), Pe-NMe(18:0/18:0)

189

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(16:0/18:1(11Z)), PC(16:0/18:1(9Z)), PC(16:1(9Z)/18:0), PC(18:0/16:1(9Z)), C42H82NO8P M+Na PC(18:1(11Z)/16:0), PC(18:1(9Z)/16:0), PC(20:0/14:1(9Z)), PC(20:1(11Z)/14:0), PE(15:0/22:1(13Z)), PE(22:1(13Z)/15:0)

PC(14:0/20:2(11Z,14Z)), PC(14:1(9Z)/20:1(11Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(16:1(9Z)/18:1(9Z)), PC(18:1(11Z)/16:1(9Z)), C42H80NO8P M+Na PC(18:1(9Z)/16:1(9Z)), PC(18:2(9Z,12Z)/16:0), PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PE(15:0/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/15:0), Pe- NMe(18:1(9Z)/18:1(9Z))

190

191

192

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(16:0/18:1(11Z)), PC(16:0/18:1(9Z)), PC(16:1(9Z)/18:0), PC(18:0/16:1(9Z)), C42H82NO8P M+H PC(18:1(11Z)/16:0), PC(18:1(9Z)/16:0), PC(20:0/14:1(9Z)), PC(20:1(11Z)/14:0), PE(15:0/22:1(13Z)), PE(22:1(13Z)/15:0)

193

PC(14:0/20:2(11Z,14Z)), PC(14:1(9Z)/20:1(11Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(16:1(9Z)/18:1(9Z)), PC(18:1(11Z)/16:1(9Z)), C42H80NO8P M+H PC(18:1(9Z)/16:1(9Z)), PC(18:2(9Z,12Z)/16:0), PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PE(15:0/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/15:0), Pe- NMe(18:1(9Z)/18:1(9Z)) C40H77NO10 M+Na Araliacerebroside PC(15:0/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/15:0), PE(14:0/22:2(13Z,16Z)), PE(14:1(9Z)/22:1(13Z)), PE(16:0/20:2(11Z,14Z)), PE(16:1(9Z)/20:1(11Z)), PE(18:0/18:2(9Z,12Z)), PE(18:1(11Z)/18:1(11Z)), C41H78NO8P M+H PE(18:1(11Z)/18:1(9Z)), PE(18:1(9Z)/18:1(11Z)), PE(18:1(9Z)/18:1(9Z)), PE(18:2(9Z,12Z)/18:0), PE(20:1(11Z)/16:1(9Z)), PE(20:2(11Z,14Z)/16:0), PE(22:1(13Z)/14:1(9Z)), PE(22:2(13Z,16Z)/14:0)

194

PC(14:0/18:2(9Z,12Z)), PC(14:1(9Z)/18:1(11Z)), PC(14:1(9Z)/18:1(9Z)), PC(16:1(9Z)/16:1(9Z)), C40H76NO8P M+H PC(18:1(11Z)/14:1(9Z)), PC(18:1(9Z)/14:1(9Z)), PC(18:2(9Z,12Z)/14:0), PE(15:0/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/15:0) PE(14:0/20:2(11Z,14Z)), PE(14:1(9Z)/20:1(11Z)), PE(16:0/18:2(9Z,12Z)), PE(16:1(9Z)/18:1(11Z)), C39H74NO8P M+H PE(16:1(9Z)/18:1(9Z)), PE(18:1(11Z)/16:1(9Z)), PE(18:1(9Z)/16:1(9Z)), PE(18:2(9Z,12Z)/16:0), PE(20:1(11Z)/14:1(9Z)), PE(20:2(11Z,14Z)/14:0) C40H49N5O6 M+H Jubanine A C35H42N4O6 M+Na Harderoporphyrinogen C36H60O3 M+H Momordicilin

PC(13:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), PE(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(15:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), C43H74NO8P M+H PC(17:2(9Z,12Z)/18:4(6Z,9Z,12Z,15Z)), PC(18:4(6Z,9Z,12Z,15Z)/17:2(9Z,12Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/15:1(9Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/13:0), PE(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), PE(P- 16:0/22:6(4Z,7Z,10Z,12E,16Z,19Z)(14OH))

195

PC(14:0/20:2(11Z,14Z)), Pe-NMe(18:1(9E)/18:1(9E)), Pe- NMe(18:1(9Z)/18:1(9Z)), PE(20:0/17:2(9Z,12Z)), PC(16:0/18:2(11Z,13Z)), PC(16:0/18:2(9E,11E)), PC(16:0/18:2(9E,11Z)), PC(16:0/18:2(9E,12E)), PC(16:1(2Z)/18:1(9Z)), PC(16:1(9Z)/18:1(9Z)), PC(17:1(9Z)/17:1(9Z)), PC(18:0/16:2(2E,4E)), PC(18:1(9Z)/16:1(9Z)), PC(18:2(2E,4E)/16:0), PC(18:2(6Z,9Z)/16:0), PC(12:0/22:2(13Z,16Z)), PC(14:1(9Z)/20:1(11Z)), PC(17:0/17:2(9Z,12Z)), PC(17:2(9Z,12Z)/17:0), PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PC(18:1(11Z)/16:1(9Z)), C42H80NO8P M+H PE(15:0/22:2(13Z,16Z)), PE(17:0/20:2(11Z,14Z)), PE(19:1(9Z)/18:1(9Z)), PE(20:1(11Z)/17:1(9Z)), PE(20:2(11Z,14Z)/17:0), PE(22:1(11Z)/15:1(9Z)), PE(22:2(13Z,16Z)/15:0), PC(16:0/18:2(10E,12Z)), PC(16:0/18:2(2E,4E)), PC(16:0/18:2(2Z,4Z)), PC(16:0/18:2(6Z,9Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(17:1(10Z)/17:1(10Z)), PC(18:2(9Z,12Z)/16:0), PC(15:1(9Z)/19:1(9Z)), PC(19:1(9Z)/15:1(9Z)), PC(22:2(13Z,16Z)/12:0), PE(15:1(9Z)/22:1(11Z)), PE(17:1(9Z)/20:1(11Z)), PE(17:2(9Z,12Z)/20:0), PE(18:1(9Z)/19:1(9Z)), PE(18:2(9Z,12Z)/19:0), PE(19:0/18:2(9Z,12Z))

196

PC(14:0/22:2(13Z,16Z)), PC(19:0/17:2(9Z,12Z)), PE(19:1(9Z)/20:1(11Z)), PC(18:1(17Z)/18:1(17Z)), PC(18:0/18:2(6Z,9Z)), PC(18:1(11E)/18:1(11E)), PC(18:1(13Z)/18:1(13Z)), PC(18:1(14Z)/18:1(14Z)), PC(18:1(15Z)/18:1(15Z)), PC(18:1(16Z)/18:1(16Z)), PC(18:1(2Z)/18:1(2Z)), PC(18:1(5Z)/18:1(5Z)), PC(18:1(9Z)/18:1(9Z)), PC(18:1(4Z)/18:1(4Z)), PC(14:1(9Z)/22:1(11Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(17:2(9Z,12Z)/19:0), PC(19:1(9Z)/17:1(9Z)), PC(20:1(11Z)/16:1(9Z)), PC(22:1(11Z)/14:1(9Z)), PC(22:1(13Z)/14:1(9Z)), PE(20:1(11Z)/19:1(9Z)), PE(21:0/18:2(9Z,12Z)), C44H84NO8P M+H PE(22:0/17:2(9Z,12Z)), PC(18:0/18:2(10Z,12Z)), PC(18:0/18:2(2E,4E)), PC(18:0/18:2(9Z,12Z)), PC(18:1(10Z)/18:1(10Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(12Z)/18:1(12Z)), PC(18:1(3Z)/18:1(3Z)), PC(18:1(6E)/18:1(6E)), PC(18:1(6Z)/18:1(6Z)), PC(18:1(7Z)/18:1(7Z)), PC(18:1(8Z)/18:1(8Z)), PC(18:1(9E)/18:1(9E)), PC(18:2(9Z,12Z)/18:0), PC(17:1(9Z)/19:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:2(13Z,16Z)/14:0), PC(14:1(9Z)/22:1(13Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PE(17:0/22:2(13Z,16Z)), PE(17:1(9Z)/22:1(11Z)), PE(17:2(9Z,12Z)/22:0), PE(18:2(9Z,12Z)/21:0), PE(19:0/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/19:0)

C42H81NO2 M+H Cer(m18:1(4E)/24:1(15Z))

PA(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PA(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PA(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PA(20:3(8Z,11Z,14Z)/20:2(11Z,14Z)), PA(22:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PA(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), PA(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), C43H75O8P M+K PA(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PA(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), PA(18:4(6Z,9Z,12Z,15Z)/22:1(11Z)), PA(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), PA(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), PA(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PA(22:2(13Z,16Z)/18:3(6Z,9Z,12Z))

197

PC(14:0/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:4(5E,8E,11E,14E)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PC(18:0/18:4(9E,11E,13E,15E)), PC(18:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(6Z,9Z)/18:2(6Z,9Z)), PC(18:2(9Z,11Z)/18:2(9Z,11Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(16:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/16:1(9Z)), PE(17:0/22:4(7Z,10Z,13Z,16Z)), PE(19:1(9Z)/20:3(8Z,11Z,14Z)), PE(22:4(7Z,10Z,13Z,16Z)/17:0), PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(18:2(2E,4E)/18:2(2E,4E)), PC(18:2(2Z,4Z)/18:2(2Z,4Z)), C44H80NO8P M+H PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PC(20:4(8E,11E,14E,17E)/16:0), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(16:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/18:3(6Z,9Z,12Z)), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PE(17:2(9Z,12Z)/22:2(13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/21:0), PE(19:0/20:4(5Z,8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/19:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/19:0), PE(21:0/18:4(6Z,9Z,12Z,15Z)), PE(22:2(13Z,16Z)/17:2(9Z,12Z))

198

PC(15:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PC(17:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:4(6Z,9Z,12Z,15Z)/19:1(9Z)), PC(19:1(9Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/17:0), PC(22:4(7Z,10Z,13Z,16Z)/15:1(9Z)), PE(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:1(11Z)), PE(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), C45H80NO8P M+H PE(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), PC(17:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PC(17:2(9Z,12Z)/20:3(8Z,11Z,14Z)), PC(20:3(8Z,11Z,14Z)/17:2(9Z,12Z)), PC(20:4(5Z,8Z,11Z,14Z)/17:1(9Z)), PE(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PE(20:3(8Z,11Z,14Z)/20:2(11Z,14Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PE(22:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:2(13Z,16Z)/18:3(9Z,12Z,15Z))

199

Table B.4B. Lipid IDs, from shotgun analyses, putatively (based on MS1 only) annotated using METASPACE software. shotgun formula adduct molecular name PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z)), PC(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/18:3(9Z,12Z,15Z)), PC(24:5(6Z,9Z,12Z,15Z,18Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:4(6Z,9Z,12Z,15Z)/24:5(6Z,9Z,12Z,15Z,18Z)), PC(24:5(9Z,12Z,15Z,18Z,21Z)/18:4(6Z,9Z,12Z,15Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(11Z,14Z,17Z)), C50H82NO8P M+H PC(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:3(11Z,14Z,17Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:4(6Z,9Z,12Z,15Z)/24:5(9Z,12Z,15Z,18Z,21Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:3(9Z,12Z,15Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PC(22:4(7Z,10Z,13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:3(6Z,9Z,12Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PC(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/18:3(6Z,9Z,12Z)) C44H83NO13 M+Na LacCer(d20:1(4E)/12:0), LacCer(d18:1(4E)/14:0), LacCer(d18:0/14:1(9Z)) C42H84NO12P M+Na IPC(t20:0/16:0), IPC(d18:0/18:0(2OH)), IPC(t18:0/18:0), IPC(d20:0/16:0(2OH)) SM(d20:0/24:4(9Z,12Z,15Z,18Z)), C49H93N2O6P M+H SM(d18:0/26:4(11Z,14Z,17Z,20Z)), SM(d16:0/28:4(13Z,16Z,19Z,22Z)), SM(d14:0/30:4(15Z,18Z,21Z,24Z))

200

PC(20:3(11Z,14Z,17Z)/18:1(9Z)), PC(34:4(19Z,22Z,25Z,28Z)/4:0), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PE(20:4(5Z,8Z,11Z,14Z)/21:0), PE(3:0/38:4(23Z,26Z,29Z,32Z)), PC(20:3(11Z,14Z,17Z)/18:1(6Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PC(16:1(9Z)/22:3(10Z,13Z,16Z)), PC(12:0/26:4(11Z,14Z,17Z,20Z)), PC(22:3(10Z,13Z,16Z)/16:1(9Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(4:0/34:4(19Z,22Z,25Z,28Z)), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PE(13:0/28:4(13Z,16Z,19Z,22Z)), PE(38:4(23Z,26Z,29Z,32Z)/3:0), PE(24:4(9Z,12Z,15Z,18Z)/17:0), PE(21:0/20:4(5Z,8Z,11Z,14Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(16:2(9Z,12Z)/22:2(13Z,16Z)), PE(21:0/20:4(8Z,11Z,14Z,17Z)), PC(18:2(9Z,11E)/20:2(11Z,14Z)), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PE(24:4(9Z,12Z,15Z,18Z)/17:0), C46H84NO8P M+Na PE(15:0/26:4(11Z,14Z,17Z,20Z)), PC(22:3(10Z,13Z,16Z)/16:1(6Z)), PC(22:4(7Z,10Z,13Z,16Z)/16:0), PC(18:1(9Z)/20:3(11Z,14Z,17Z)), PE(28:4(13Z,16Z,19Z,22Z)/13:0), PE(22:4(7Z,10Z,13Z,16Z)/19:0), PE(19:0/22:4(7Z,10Z,13Z,16Z)), PC(26:4(11Z,14Z,17Z,20Z)/12:0), PC(20:2(11Z,14Z)/18:2(9Z,11E)), PC(28:4(13Z,16Z,19Z,22Z)/10:0), PC(22:2(13Z,16Z)/16:2(9Z,12Z)), PC(36:4(21Z,24Z,27Z,30Z)/2:0), PC(20:3(8Z,11Z,14Z)/18:1(6Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PE(26:4(11Z,14Z,17Z,20Z)/15:0), PC(18:1(11E)/20:3(11Z,14Z,17Z)), PC(30:4(15Z,18Z,21Z,24Z)/8:0), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(16:1(6Z)/22:3(10Z,13Z,16Z)), PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(20:3(11Z,14Z,17Z)/18:1(11Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(18:3(9Z,12Z,15Z)/20:1(11Z)),

201

PC(20:3(11Z,14Z,17Z)/18:1(11E)), PC(20:4(8Z,11Z,14Z,17Z)/18:0), PC(8:0/30:4(15Z,18Z,21Z,24Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z))

C44H81NO13 M+H LacCer(d18:1(4E)/14:1(9Z))

202

PC(20:0/16:2(9Z,12Z)), PC(16:0/20:2(11Z,14Z)), PC(18:1(9Z)/18:1(9Z)), PC(20:1(11Z)/16:1(9Z)), PC(18:1(11Z)/18:1(9Z)), PC(16:2(9Z,12Z)/20:0), PE(18:2(9Z,11E)/21:0), PC(20:2(11Z,14Z)/16:0), PE(21:0/18:2(9Z,11E)), PC(18:1(11E)/18:1(6Z)), PC(18:1(11E)/18:1(9Z)), PE(22:2(13Z,16Z)/17:0), PC(18:1(11E)/18:1(11Z)), PE(17:0/22:2(13Z,16Z)), PE(17:0/22:2(13Z,16Z)), PC(18:2(9Z,11E)/18:0), PC(16:1(9Z)/20:1(11Z)), PE(22:2(13Z,16Z)/17:0), C44H84NO8P M+K PE(21:0/18:2(9Z,12Z)), PC(16:1(6Z)/20:1(11Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:1(6Z)/18:1(6Z)), PE(20:2(11Z,14Z)/19:0), PC(20:1(11Z)/16:1(6Z)), PC(22:1(13Z)/14:1(9Z)), PC(18:1(6Z)/18:1(9Z)), PE(19:0/20:2(11Z,14Z)), PC(18:1(11Z)/18:1(6Z)), PC(18:0/18:2(9Z,11E)), PC(14:0/22:2(13Z,16Z)), PC(18:1(6Z)/18:1(11E)), PC(18:1(6Z)/18:1(11Z)), PC(18:0/18:2(9Z,12Z)), PC(18:1(11Z)/18:1(11E)), PC(18:1(9Z)/18:1(11E)), PC(18:1(11E)/18:1(11E)), PC(22:2(13Z,16Z)/14:0), PE(18:2(9Z,12Z)/21:0), PC(18:2(9Z,12Z)/18:0), PC(18:1(9Z)/18:1(6Z)), PC(14:1(9Z)/22:1(13Z)), PC(18:1(11Z)/18:1(11Z))

PE(P-18:1(11Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(P- 18:1(9Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(O- 18:2(9Z,12Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(P- C47H80NO7P M+Na 20:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(P- 18:2(9Z,12Z)/24:5(9Z,12Z,15Z,18Z,21Z)), PE(P- 18:1(13Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(P- 18:2(9Z,12Z)/24:5(6Z,9Z,12Z,15Z,18Z))

203

PG(18:1(6Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Lysobisphosphatidate (22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0/18:1(11E)/0:0), PG(18:1(11E)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PG(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), PG(24:5(9Z,12Z,15Z,18Z,21Z)/16:2(9Z,12Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PG(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), PG(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Lysobisphosphatidate (20:3(11Z,14Z,17Z)/0:0/20:4(8Z,11Z,14Z,17Z)/0:0), Lysobisphosphatidate (18:4(6Z,9Z,12Z,15Z)/0:0/22:3(10Z,13Z,16Z)/0:0), Lysobisphosphatidate (24:5(9Z,12Z,15Z,18Z,21Z)/0:0/16:2(9Z,12Z)/0:0), PG(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,11E)), PG(26:6(8Z,11Z,14Z,17Z,20Z,23Z)/14:1(9Z)), Lysobisphosphatidate (20:5(5Z,8Z,11Z,14Z,17Z)/0:0/20:2(11Z,14Z)/0:0), Lysobisphosphatidate (24:6(6Z,9Z,12Z,15Z,18Z,21Z)/0:0/16:1(6Z)/0:0), PG(18:4(6Z,9Z,12Z,15Z)/22:3(10Z,13Z,16Z)), C46H77O10P M+H PG(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PG(16:1(9Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PG(14:1(9Z)/26:6(8Z,11Z,14Z,17Z,20Z,23Z)), PG(16:1(6Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PG(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), PG(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), PG(20:3(8Z,11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Lysobisphosphatidate (26:6(8Z,11Z,14Z,17Z,20Z,23Z)/0:0/14:1(9Z)/0:0), Lysobisphosphatidate (18:3(9Z,12Z,15Z)/0:0/22:4(7Z,10Z,13Z,16Z)/0:0), Lysobisphosphatidate (22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0/18:1(11Z)/0:0), Lysobisphosphatidate (24:6(6Z,9Z,12Z,15Z,18Z,21Z)/0:0/16:1(9Z)/0:0), PG(20:3(11Z,14Z,17Z)/20:4(5Z,8Z,11Z,14Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:3(11Z,14Z,17Z)), PG(18:2(9Z,11E)/22:5(7Z,10Z,13Z,16Z,19Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PG(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/16:1(6Z)), PG(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), PG(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Lysobisphosphatidate

204

(22:5(7Z,10Z,13Z,16Z,19Z)/0:0/18:2(9Z,12Z)/0:0), PG(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Lysobisphosphatidate (20:3(11Z,14Z,17Z)/0:0/20:4(5Z,8Z,11Z,14Z)/0:0), Lysobisphosphatidate (18:3(6Z,9Z,12Z)/0:0/22:4(7Z,10Z,13Z,16Z)/0:0), Lysobisphosphatidate (22:5(4Z,7Z,10Z,13Z,16Z)/0:0/18:2(9Z,11E)/0:0), PG(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11E)), PG(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,11E)), PG(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PG(20:4(8Z,11Z,14Z,17Z)/20:3(11Z,14Z,17Z)), Lysobisphosphatidate (16:2(9Z,12Z)/0:0/24:5(6Z,9Z,12Z,15Z,18Z)/0:0), Lysobisphosphatidate (20:3(8Z,11Z,14Z)/0:0/20:4(5Z,8Z,11Z,14Z)/0:0), PG(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/16:1(9Z)), Lysobisphosphatidate (20:4(8Z,11Z,14Z,17Z)/0:0/20:3(8Z,11Z,14Z)/0:0), Lysobisphosphatidate (22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0/18:1(9Z)/0:0)

205

PC(20:3(11Z,14Z,17Z)/18:1(9Z)), PC(34:4(19Z,22Z,25Z,28Z)/4:0), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PE(20:4(5Z,8Z,11Z,14Z)/21:0), PE(3:0/38:4(23Z,26Z,29Z,32Z)), PC(20:3(11Z,14Z,17Z)/18:1(6Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PC(16:1(9Z)/22:3(10Z,13Z,16Z)), PC(12:0/26:4(11Z,14Z,17Z,20Z)), PC(22:3(10Z,13Z,16Z)/16:1(9Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(4:0/34:4(19Z,22Z,25Z,28Z)), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PE(13:0/28:4(13Z,16Z,19Z,22Z)), PE(38:4(23Z,26Z,29Z,32Z)/3:0), PE(24:4(9Z,12Z,15Z,18Z)/17:0), PE(21:0/20:4(5Z,8Z,11Z,14Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(16:2(9Z,12Z)/22:2(13Z,16Z)), PE(21:0/20:4(8Z,11Z,14Z,17Z)), PC(18:2(9Z,11E)/20:2(11Z,14Z)), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PE(24:4(9Z,12Z,15Z,18Z)/17:0), C46H84NO8P M+H PE(15:0/26:4(11Z,14Z,17Z,20Z)), PC(22:3(10Z,13Z,16Z)/16:1(6Z)), PC(22:4(7Z,10Z,13Z,16Z)/16:0), PC(18:1(9Z)/20:3(11Z,14Z,17Z)), PE(28:4(13Z,16Z,19Z,22Z)/13:0), PE(22:4(7Z,10Z,13Z,16Z)/19:0), PE(19:0/22:4(7Z,10Z,13Z,16Z)), PC(26:4(11Z,14Z,17Z,20Z)/12:0), PC(20:2(11Z,14Z)/18:2(9Z,11E)), PC(28:4(13Z,16Z,19Z,22Z)/10:0), PC(22:2(13Z,16Z)/16:2(9Z,12Z)), PC(36:4(21Z,24Z,27Z,30Z)/2:0), PC(20:3(8Z,11Z,14Z)/18:1(6Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PE(26:4(11Z,14Z,17Z,20Z)/15:0), PC(18:1(11E)/20:3(11Z,14Z,17Z)), PC(30:4(15Z,18Z,21Z,24Z)/8:0), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(16:1(6Z)/22:3(10Z,13Z,16Z)), PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(20:3(11Z,14Z,17Z)/18:1(11Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(18:3(9Z,12Z,15Z)/20:1(11Z)),

206

PC(20:3(11Z,14Z,17Z)/18:1(11E)), PC(20:4(8Z,11Z,14Z,17Z)/18:0), PC(8:0/30:4(15Z,18Z,21Z,24Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z))

207

PC(12:0/24:1(15Z)), PE(21:0/18:1(11Z)), PE(15:0/24:1(15Z)), PC(26:1(17Z)/10:0), PE(22:1(13Z)/17:0), PE(22:1(13Z)/17:0), PE(21:0/18:1(11E)), PC(14:0/22:1(13Z)), PE(18:1(6Z)/21:0), PE(17:0/22:1(13Z)), PC(16:1(6Z)/20:0), PE(17:0/22:1(13Z)), C44H86NO8P M+Na PE(26:1(17Z)/13:0), PC(22:1(13Z)/14:0), PC(14:1(9Z)/22:0), PE(21:0/18:1(6Z)), PC(20:0/16:1(6Z)), PC(18:0/18:1(11E)), PE(13:0/26:1(17Z)), PE(19:0/20:1(11Z)), PC(24:1(15Z)/12:0), PC(18:0/18:1(11Z)), PC(20:1(11Z)/16:0), PE(21:0/18:1(9Z)), PC(22:0/14:1(9Z)), PC(18:1(11Z)/18:0), PE(18:1(11E)/21:0), PE(24:1(15Z)/15:0), PC(10:0/26:1(17Z)), PE(24:1(15Z)/15:0), PC(18:1(11E)/18:0), PC(18:0/18:1(6Z)), PE(18:1(11Z)/21:0), PE(15:0/24:1(15Z)), PE(26:1(17Z)/13:0), PC(18:1(6Z)/18:0), PC(16:0/20:1(11Z)), PE(13:0/26:1(17Z)), PC(16:1(9Z)/20:0), PE(20:1(11Z)/19:0), PE(18:1(9Z)/21:0), PC(18:0/18:1(9Z)), PC(18:1(9Z)/18:0), PC(20:0/16:1(9Z)) C42H81NO13 M+H LacCer(d18:0/12:0), LacCer(d20:0/10:0)

208

PC(14:1(9Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PC(18:3(9Z,12Z,15Z)/20:4(5Z,8Z,11Z,14Z)), PC(16:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:3(6Z,9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:3(9Z,12Z,15Z)), PC(20:3(11Z,14Z,17Z)/18:4(6Z,9Z,12Z,15Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:3(11Z,14Z,17Z)), PC(16:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/16:2(9Z,12Z)), PC(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/14:1(9Z)), C46H78NO8P M+H PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:1(6Z)), PC(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:2(9Z,11E)), PC(16:1(6Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/16:2(9Z,12Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PC(18:3(6Z,9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:3(6Z,9Z,12Z)), PC(18:2(9Z,11E)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), PC(20:3(8Z,11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PC(18:3(9Z,12Z,15Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z))

209

PC(20:3(11Z,14Z,17Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:2(9Z,12Z)), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(18:3(9Z,12Z,15Z)/18:1(11E)), PC(18:2(9Z,11E)/18:2(9Z,11E)), PE(13:0/26:4(11Z,14Z,17Z,20Z)), PE(36:4(21Z,24Z,27Z,30Z)/3:0), PE(20:4(5Z,8Z,11Z,14Z)/19:0), PE(24:4(9Z,12Z,15Z,18Z)/15:0), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(20:3(8Z,11Z,14Z)/16:1(6Z)), PC(18:2(9Z,12Z)/18:2(9Z,11E)), PC(2:0/34:4(19Z,22Z,25Z,28Z)), PC(18:1(11E)/18:3(6Z,9Z,12Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:3(6Z,9Z,12Z)/18:1(11E)), PC(16:1(6Z)/20:3(11Z,14Z,17Z)), PC(18:1(6Z)/18:3(9Z,12Z,15Z)), PC(12:0/24:4(9Z,12Z,15Z,18Z)), PE(15:0/24:4(9Z,12Z,15Z,18Z)), PC(34:4(19Z,22Z,25Z,28Z)/2:0), C44H80NO8P M+Na PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(18:3(9Z,12Z,15Z)/18:1(6Z)), PC(4:0/32:4(17Z,20Z,23Z,26Z)), PE(18:4(6Z,9Z,12Z,15Z)/21:0), PC(32:4(17Z,20Z,23Z,26Z)/4:0), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PE(22:4(7Z,10Z,13Z,16Z)/17:0), PE(21:0/18:4(6Z,9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PE(20:4(8Z,11Z,14Z,17Z)/19:0), PE(13:0/26:4(11Z,14Z,17Z,20Z)), PC(20:3(11Z,14Z,17Z)/16:1(6Z)), PC(16:2(9Z,12Z)/20:2(11Z,14Z)), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PE(24:4(9Z,12Z,15Z,18Z)/15:0), PE(19:0/20:4(5Z,8Z,11Z,14Z)), PC(18:2(9Z,11E)/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(10:0/26:4(11Z,14Z,17Z,20Z)), PE(17:0/22:4(7Z,10Z,13Z,16Z)), PC(18:1(6Z)/18:3(6Z,9Z,12Z)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PE(3:0/36:4(21Z,24Z,27Z,30Z)), PC(22:3(10Z,13Z,16Z)/14:1(9Z)),

210

PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(16:1(9Z)/20:3(11Z,14Z,17Z))

C40H80NO12P M+H IPC(d18:0/16:0(2OH)), IPC(d20:0/14:0(2OH)), IPC(t18:0/16:0), IPC(t20:0/14:0) C45H77NO9 M+Na GlcCer(iso-t17:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))

211

PC(18:1(9Z)/16:0), PE(18:1(9Z)/19:0), PE(19:0/18:1(9Z)), PC(16:0/18:1(9Z)), PC(20:0/14:1(9Z)), PE(24:1(15Z)/13:0), PE(15:0/22:1(13Z)), PC(26:1(17Z)/8:0), PC(18:1(6Z)/16:0), PE(20:1(11Z)/17:0), PE(13:0/24:1(15Z)), PC(20:1(11Z)/14:0), PC(16:1(9Z)/18:0), PE(17:0/20:1(11Z)), PC(24:1(15Z)/10:0), PE(24:1(15Z)/13:0), PE(21:0/16:1(9Z)), PC(16:0/18:1(11Z)), C42H82NO8P M+K PE(18:1(11E)/19:0), PE(19:0/18:1(6Z)), PE(22:1(13Z)/15:0), PC(22:1(13Z)/12:0), PC(18:0/16:1(9Z)), PC(8:0/26:1(17Z)), PE(20:1(11Z)/17:0), PE(22:1(13Z)/15:0), PE(16:1(9Z)/21:0), PC(16:0/18:1(6Z)), PC(18:1(11E)/16:0), PC(14:1(9Z)/20:0), PC(12:0/22:1(13Z)), PC(14:0/20:1(11Z)), PE(18:1(11Z)/19:0), PC(18:0/16:1(6Z)), PE(13:0/24:1(15Z)), PE(21:0/16:1(6Z)), PE(19:0/18:1(11Z)), PC(16:0/18:1(11E)), PE(15:0/22:1(13Z)), PE(18:1(6Z)/19:0), PC(18:1(11Z)/16:0), PE(19:0/18:1(11E)), PC(16:1(6Z)/18:0), PE(16:1(6Z)/21:0), PE(17:0/20:1(11Z)), PC(10:0/24:1(15Z))

PE(P-20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(P- 16:0/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(P- 18:1(13Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(P- 14:0/26:6(8Z,11Z,14Z,17Z,20Z,23Z)), PE(O- 18:1(13Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(P- 15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(P- 18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(O- C45H78NO7P M+Na 18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(P- 18:1(13Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(P- 18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(P- 18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PE(P- 18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(O- 18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(P- 13:0/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(O- 18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(O- 18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(P- 18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(P- 18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z))

212

PG(18:1(11Z)/18:1(9Z)), PG(18:1(6Z)/18:1(9Z)), PG(18:2(9Z,11E)/18:0), PG(18:1(11Z)/18:1(11Z)), PG(20:1(11Z)/16:1(9Z)), PG(18:1(11E)/18:1(11E)), PG(16:0/20:2(11Z,14Z)), PG(18:1(11E)/18:1(9Z)), Lysobisphosphatidate (18:0/0:0/18:2(9Z,11E)/0:0), Lysobisphosphatidate (16:1(9Z)/0:0/20:1(11Z)/0:0), Lysobisphosphatidate (18:1(11E)/0:0/18:1(6Z)/0:0), PG(20:0/16:2(9Z,12Z)), Bis-[2-(9Z-octadecenoyl)-sn-glycero]- 1-phosphate, Lysobisphosphatidate (18:1(11Z)/0:0/18:1(9Z)/0:0), Lysobisphosphatidate (18:1(11Z)/0:0/18:1(6Z)/0:0), PG(22:1(13Z)/14:1(9Z)), PG(18:2(9Z,12Z)/18:0), PG(18:1(9Z)/18:1(11Z)), PG(18:1(9Z)/18:1(6Z)), Lysobisphosphatidate (18:0/0:0/18:2(9Z,12Z)/0:0), Bis-[2-(11E-octadecenoyl)-sn- glycero]-1-phosphate, PG(16:1(6Z)/20:1(11Z)), PG(18:0/18:2(9Z,12Z)), PG(14:0/22:2(13Z,16Z)), PG(16:2(9Z,12Z)/20:0), PG(20:1(11Z)/16:1(6Z)), C42H79O10P M+Na PG(16:1(9Z)/20:1(11Z)), PG(14:1(9Z)/22:1(13Z)), Lysobisphosphatidate (14:0/0:0/22:2(13Z,16Z)/0:0), Bis-[2-(6Z- octadecenoyl)-sn-glycero]-1-phosphate, PG(18:1(11Z)/18:1(6Z)), Lysobisphosphatidate (16:0/0:0/20:2(11Z,14Z)/0:0), Lysobisphosphatidate (20:0/0:0/16:2(9Z,12Z)/0:0), Lysobisphosphatidate (18:1(11Z)/0:0/18:1(11E)/0:0), Lysobisphosphatidate (18:1(11E)/0:0/18:1(9Z)/0:0), Lysobisphosphatidate (22:1(13Z)/0:0/14:1(9Z)/0:0), PG(18:1(6Z)/18:1(11Z)), PG(18:1(9Z)/18:1(11E)), PG(18:0/18:2(9Z,11E)), Bis-[2-(11Z- octadecenoyl)-sn-glycero]-1-phosphate, PG(18:1(11E)/18:1(11Z)), PG(20:2(11Z,14Z)/16:0), PG(22:2(13Z,16Z)/14:0), PG(18:1(6Z)/18:1(6Z)), PG(18:1(9Z)/18:1(9Z)), PG(18:1(11E)/18:1(6Z)), PG(18:1(11Z)/18:1(11E)), PG(18:1(6Z)/18:1(11E)), Lysobisphosphatidate (18:1(9Z)/0:0/18:1(6Z)/0:0), Lysobisphosphatidate (20:1(11Z)/0:0/16:1(6Z)/0:0)

213

PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:3(11Z,14Z,17Z)), PE(22:3(10Z,13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:3(11Z,14Z,17Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), PE(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,11E)), PE(24:5(6Z,9Z,12Z,15Z,18Z)/16:2(9Z,12Z)), PE(18:1(6Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,11E)), PE(16:1(9Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(20:3(11Z,14Z,17Z)/20:4(5Z,8Z,11Z,14Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11E)), PE(16:1(6Z)/24:6(6Z,9Z,12Z,15Z,18Z,21Z)), PE(16:2(9Z,12Z)/24:5(6Z,9Z,12Z,15Z,18Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PE(26:6(8Z,11Z,14Z,17Z,20Z,23Z)/14:1(9Z)), PE(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:1(11E)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), C45H76NO8P M+H PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(6Z)), PE(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PE(16:2(9Z,12Z)/24:5(9Z,12Z,15Z,18Z,21Z)), PE(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:3(11Z,14Z,17Z)), PE(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PE(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/16:1(6Z)), PE(20:3(8Z,11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), PE(24:5(9Z,12Z,15Z,18Z,21Z)/16:2(9Z,12Z)), PE(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(24:6(6Z,9Z,12Z,15Z,18Z,21Z)/16:1(9Z)), PE(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:3(10Z,13Z,16Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:3(8Z,11Z,14Z)), PE(18:2(9Z,11E)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:2(9Z,11E)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(14:1(9Z)/26:6(8Z,11Z,14Z,17Z,20Z,23Z))

214

PC(18:4(6Z,9Z,12Z,15Z)/17:0), PC(15:0/20:4(8Z,11Z,14Z,17Z)), PE(2:0/36:4(21Z,24Z,27Z,30Z)), PC(13:0/22:4(7Z,10Z,13Z,16Z)), PC(20:4(8Z,11Z,14Z,17Z)/15:0), PE(16:2(9Z,12Z)/22:2(13Z,16Z)), PC(20:4(5Z,8Z,11Z,14Z)/15:0), PC(22:4(7Z,10Z,13Z,16Z)/13:0), PE(20:1(11Z)/18:3(9Z,12Z,15Z)), PE(18:1(6Z)/20:3(11Z,14Z,17Z)), PE(18:0/20:4(8Z,11Z,14Z,17Z)), PC(15:0/20:4(5Z,8Z,11Z,14Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:0), PE(20:0/18:4(6Z,9Z,12Z,15Z)), PC(17:0/18:4(6Z,9Z,12Z,15Z)), PE(30:4(15Z,18Z,21Z,24Z)/8:0), PE(36:4(21Z,24Z,27Z,30Z)/2:0), PE(16:1(6Z)/22:3(10Z,13Z,16Z)), PE(20:3(8Z,11Z,14Z)/18:1(11E)), PE(24:4(9Z,12Z,15Z,18Z)/14:0), PE(18:1(11E)/20:3(8Z,11Z,14Z)), PE(20:2(11Z,14Z)/18:2(9Z,11E)), PE(18:1(11Z)/20:3(11Z,14Z,17Z)), PE(32:4(17Z,20Z,23Z,26Z)/6:0), PE(20:3(8Z,11Z,14Z)/18:1(9Z)), C43H78NO8P M+Na PE(12:0/26:4(11Z,14Z,17Z,20Z)), PC(20:4(8Z,11Z,14Z,17Z)/15:0), PE(20:1(11Z)/18:3(6Z,9Z,12Z)), PE(18:2(9Z,12Z)/20:2(11Z,14Z)), PE(4:0/34:4(19Z,22Z,25Z,28Z)), PE(16:0/22:4(7Z,10Z,13Z,16Z)), PC(15:0/20:4(8Z,11Z,14Z,17Z)), PE(22:3(10Z,13Z,16Z)/16:1(9Z)), PE(18:1(11E)/20:3(11Z,14Z,17Z)), PE(18:1(11Z)/20:3(8Z,11Z,14Z)), PE(28:4(13Z,16Z,19Z,22Z)/10:0), PC(18:4(6Z,9Z,12Z,15Z)/17:0), PC(22:4(7Z,10Z,13Z,16Z)/13:0), PC(3:0/32:4(17Z,20Z,23Z,26Z)), PE(26:4(11Z,14Z,17Z,20Z)/12:0), PE(20:3(11Z,14Z,17Z)/18:1(11Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:0), PC(13:0/22:4(7Z,10Z,13Z,16Z)), PE(22:2(13Z,16Z)/16:2(9Z,12Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:0), PC(32:4(17Z,20Z,23Z,26Z)/3:0), PE(18:1(6Z)/20:3(8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/18:1(11Z)),

215

PE(8:0/30:4(15Z,18Z,21Z,24Z)), PE(22:3(10Z,13Z,16Z)/16:1(6Z))

216

PC(20:3(11Z,14Z,17Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:2(9Z,12Z)), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(18:3(9Z,12Z,15Z)/18:1(11E)), PC(18:2(9Z,11E)/18:2(9Z,11E)), PE(13:0/26:4(11Z,14Z,17Z,20Z)), PE(36:4(21Z,24Z,27Z,30Z)/3:0), PE(20:4(5Z,8Z,11Z,14Z)/19:0), PE(24:4(9Z,12Z,15Z,18Z)/15:0), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(20:3(8Z,11Z,14Z)/16:1(6Z)), PC(18:2(9Z,12Z)/18:2(9Z,11E)), PC(2:0/34:4(19Z,22Z,25Z,28Z)), PC(18:1(11E)/18:3(6Z,9Z,12Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:3(6Z,9Z,12Z)/18:1(11E)), PC(16:1(6Z)/20:3(11Z,14Z,17Z)), PC(18:1(6Z)/18:3(9Z,12Z,15Z)), PC(12:0/24:4(9Z,12Z,15Z,18Z)), PE(15:0/24:4(9Z,12Z,15Z,18Z)), PC(34:4(19Z,22Z,25Z,28Z)/2:0), C44H80NO8P M+H PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(18:3(9Z,12Z,15Z)/18:1(6Z)), PC(4:0/32:4(17Z,20Z,23Z,26Z)), PE(18:4(6Z,9Z,12Z,15Z)/21:0), PC(32:4(17Z,20Z,23Z,26Z)/4:0), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PE(22:4(7Z,10Z,13Z,16Z)/17:0), PE(21:0/18:4(6Z,9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PE(20:4(8Z,11Z,14Z,17Z)/19:0), PE(13:0/26:4(11Z,14Z,17Z,20Z)), PC(20:3(11Z,14Z,17Z)/16:1(6Z)), PC(16:2(9Z,12Z)/20:2(11Z,14Z)), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PE(24:4(9Z,12Z,15Z,18Z)/15:0), PE(19:0/20:4(5Z,8Z,11Z,14Z)), PC(18:2(9Z,11E)/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(10:0/26:4(11Z,14Z,17Z,20Z)), PE(17:0/22:4(7Z,10Z,13Z,16Z)), PC(18:1(6Z)/18:3(6Z,9Z,12Z)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PE(3:0/36:4(21Z,24Z,27Z,30Z)), PC(22:3(10Z,13Z,16Z)/14:1(9Z)),

217

PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(16:1(9Z)/20:3(11Z,14Z,17Z))

218

PC(18:1(9Z)/16:0), PE(18:1(9Z)/19:0), PE(19:0/18:1(9Z)), PC(16:0/18:1(9Z)), PC(20:0/14:1(9Z)), PE(24:1(15Z)/13:0), PE(15:0/22:1(13Z)), PC(26:1(17Z)/8:0), PC(18:1(6Z)/16:0), PE(20:1(11Z)/17:0), PE(13:0/24:1(15Z)), PC(20:1(11Z)/14:0), PC(16:1(9Z)/18:0), PE(17:0/20:1(11Z)), PC(24:1(15Z)/10:0), PE(24:1(15Z)/13:0), PE(21:0/16:1(9Z)), PC(16:0/18:1(11Z)), C42H82NO8P M+Na PE(18:1(11E)/19:0), PE(19:0/18:1(6Z)), PE(22:1(13Z)/15:0), PC(22:1(13Z)/12:0), PC(18:0/16:1(9Z)), PC(8:0/26:1(17Z)), PE(20:1(11Z)/17:0), PE(22:1(13Z)/15:0), PE(16:1(9Z)/21:0), PC(16:0/18:1(6Z)), PC(18:1(11E)/16:0), PC(14:1(9Z)/20:0), PC(12:0/22:1(13Z)), PC(14:0/20:1(11Z)), PE(18:1(11Z)/19:0), PC(18:0/16:1(6Z)), PE(13:0/24:1(15Z)), PE(21:0/16:1(6Z)), PE(19:0/18:1(11Z)), PC(16:0/18:1(11E)), PE(15:0/22:1(13Z)), PE(18:1(6Z)/19:0), PC(18:1(11Z)/16:0), PE(19:0/18:1(11E)), PC(16:1(6Z)/18:0), PE(16:1(6Z)/21:0), PE(17:0/20:1(11Z)), PC(10:0/24:1(15Z))

PC(18:1(6Z)/16:1(9Z)), PC(16:1(9Z)/18:1(11Z)), PC(18:1(9Z)/16:1(6Z)), PE(22:2(13Z,16Z)/15:0), PE(17:0/20:2(11Z,14Z)), PE(18:2(9Z,12Z)/19:0), PC(18:1(11E)/16:1(6Z)), PE(20:2(11Z,14Z)/17:0), PC(22:2(13Z,16Z)/12:0), PC(16:1(9Z)/18:1(6Z)), PC(16:0/18:2(9Z,11E)), PC(16:0/18:2(9Z,12Z)), PC(12:0/22:2(13Z,16Z)), PC(18:1(11Z)/16:1(9Z)), PC(14:0/20:2(11Z,14Z)), PE(16:2(9Z,12Z)/21:0), C42H80NO8P M+Na PC(18:1(6Z)/16:1(6Z)), PC(18:2(9Z,12Z)/16:0), PC(16:1(9Z)/18:1(11E)), PE(21:0/16:2(9Z,12Z)), PC(16:1(6Z)/18:1(11E)), PC(16:1(9Z)/18:1(9Z)), PE(19:0/18:2(9Z,12Z)), PC(18:1(11Z)/16:1(6Z)), PC(18:2(9Z,11E)/16:0), PC(16:2(9Z,12Z)/18:0), PC(16:1(6Z)/18:1(9Z)), PC(18:1(9Z)/16:1(9Z)), PC(16:1(6Z)/18:1(6Z)), PE(17:0/20:2(11Z,14Z)), PC(18:0/16:2(9Z,12Z)), PE(20:2(11Z,14Z)/17:0), PE(18:2(9Z,11E)/19:0), PE(15:0/22:2(13Z,16Z)), PC(16:1(6Z)/18:1(11Z)), PC(14:1(9Z)/20:1(11Z)), PE(15:0/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/15:0), PC(18:1(11E)/16:1(9Z)), PE(19:0/18:2(9Z,11E)), PC(20:2(11Z,14Z)/14:0), PC(20:1(11Z)/14:1(9Z)), 1,2-di-(9Z- octadecenoyl)-sn-glycero-3-phospho-N-methylethanolamine C40H77NO13 M+H LacCer(d18:0/10:0), LacCer(d20:0/8:0)

219

PC(16:0/16:1(6Z)), PC(14:0/18:1(11Z)), PC(14:0/18:1(6Z)), PE(17:0/18:1(9Z)), PC(6:0/26:1(17Z)), PC(16:1(9Z)/16:0), PE(17:0/18:1(11Z)), PC(24:1(15Z)/8:0), PC(14:0/18:1(9Z)), PE(22:1(13Z)/13:0), PC(8:0/24:1(15Z)), PE(20:1(11Z)/15:0), PE(17:0/18:1(11E)), PE(14:1(9Z)/21:0), PE(19:0/16:1(9Z)), PC(18:1(11Z)/14:0), PC(14:1(9Z)/18:0), PC(12:0/20:1(11Z)), C40H78NO8P M+Na PE(18:1(6Z)/17:0), PE(19:0/16:1(6Z)), PC(16:1(6Z)/16:0), PC(18:1(6Z)/14:0), PC(18:1(9Z)/14:0), PE(20:1(11Z)/15:0), PE(18:1(11Z)/17:0), PE(17:0/18:1(6Z)), PE(17:0/18:1(9Z)), PE(18:1(9Z)/17:0), PE(15:0/20:1(11Z)), PE(13:0/22:1(13Z)), PE(18:1(11E)/17:0), PC(20:1(11Z)/12:0), PE(17:0/18:1(6Z)), PE(18:1(9Z)/17:0), PC(10:0/22:1(13Z)), PE(13:0/22:1(13Z)), PE(21:0/14:1(9Z)), PC(16:0/16:1(9Z)), PC(18:1(11E)/14:0), PE(18:1(6Z)/17:0), PE(17:0/18:1(11Z)), PE(18:1(11Z)/17:0), PE(16:1(6Z)/19:0), PE(16:1(9Z)/19:0), PE(17:0/18:1(11E)), PC(22:1(13Z)/10:0), PC(18:0/14:1(9Z)), PE(18:1(11E)/17:0), PE(15:0/20:1(11Z)), PE(22:1(13Z)/13:0)

SM(d18:1(4E)/18:3(9Z,12Z,15Z)), SM(d16:1(4E)/20:3(8Z,11Z,14Z)), SM(d16:0/20:4(8Z,11Z,14Z,17Z)), C41H77N2O6P M+H SM(d18:0/18:4(6Z,9Z,12Z,15Z)), SM(d18:1(4E)/18:3(6Z,9Z,12Z)), SM(d16:0/20:4(5Z,8Z,11Z,14Z)), SM(d14:0/22:4(7Z,10Z,13Z,16Z)), SM(d16:1(4E)/20:3(11Z,14Z,17Z)), SM(d14:1(4E)/22:3(10Z,13Z,16Z))

SM(iso-d17:1(4E)/17:0), SM(d18:1(4E)/16:0), PE- Cer(d14:1(4E)/23:0), SM(d14:1(4E)/20:0), SM(iso- C39H79N2O6P M+Na d17:1(4E)/17:0), SM(d20:1(4E)/14:0), SM(d14:0/20:1(11Z)), SM(d18:0/16:1(6Z)), PE-Cer(d18:1(4E)/19:0), SM(d20:0/14:1(9Z)), SM(d16:0/18:1(11E)), SM(d16:0/18:1(6Z)), SM(d16:0/18:1(9Z)), SM(d16:1(4E)/18:0), SM(d18:0/16:1(9Z)), SM(d16:0/18:1(11Z)), PE- Cer(d16:1(4E)/21:0) C68H50O44 M+Na Heterophylliin F

220

CL(i-13:0/i-22:0/18:2(9Z,11Z)/a-25:0)[rac], CL(a-13:0/i- 22:0/18:2(9Z,11Z)/a-25:0)[rac], CL(a-13:0/i-22:0/a- 25:0/18:2(9Z,11Z))[rac], CL(i-13:0/i-22:0/a- 25:0/18:2(9Z,11Z))[rac], CL(i-13:0/a-25:0/18:2(9Z,11Z)/i- 22:0)[rac], CL(a-13:0/a-25:0/18:2(9Z,11Z)/i-22:0)[rac], CL(i- C87H166O17P2 M+Na 13:0/a-25:0/i-22:0/18:2(9Z,11Z))[rac], CL(a-13:0/a-25:0/i- 22:0/18:2(9Z,11Z))[rac], CL(i-13:0/18:2(9Z,11Z)/a-25:0/i- 22:0)[rac], CL(a-13:0/18:2(9Z,11Z)/a-25:0/i-22:0)[rac], CL(i- 12:0/18:2(9Z,11Z)/i-24:0/i-24:0), CL(a-13:0/18:2(9Z,11Z)/i- 22:0/a-25:0)[rac], CL(i-13:0/18:2(9Z,11Z)/i-22:0/a-25:0)[rac], CL(i-12:0/i-24:0/18:2(9Z,11Z)/i-24:0), CL(i-12:0/i-24:0/i- 24:0/18:2(9Z,11Z)) C68H44O44 M+H Punicalin TG(16:0/18:2(9Z,12Z)/20:1(11Z)), TG(18:0/18:1(9Z)/18:2(9Z,12Z)), TG(16:1(9Z)/18:1(9Z)/20:1(11Z)), TG(16:1(9Z)/18:2(9Z,12Z)/20:0), TG(18:1(9Z)/18:1(9Z)/18:1(9Z)), TG(18:1(11Z)/18:0/18:2(9Z,12Z)), TG(18:1(9Z)/16:0/20:2(11Z,14Z)), TG(18:1(9Z)/18:1(11Z)/18:1(9Z)), Tripetroselinin, TG(14:0/20:0/20:3(5Z,8Z,11Z)), TG(14:0/20:0/20:3n6), TG(14:0/22:0/18:3(6Z,9Z,12Z)), TG(14:0/22:0/18:3(9Z,12Z,15Z)), TG(14:0/18:1(11Z)/22:2(13Z,16Z)), TG(14:0/18:1(9Z)/22:2(13Z,16Z)), TG(14:0/20:1(11Z)/20:2n6), TG(14:0/20:3(5Z,8Z,11Z)/20:0), TG(14:0/22:1(13Z)/18:2(9Z,12Z)), C57H104O6 M+Na TG(14:0/18:2(9Z,12Z)/22:1(13Z)), TG(14:0/18:3(6Z,9Z,12Z)/22:0), TG(14:0/20:2n6/20:1(11Z)), TG(14:0/20:3n6/20:0), TG(14:0/22:2(13Z,16Z)/18:1(11Z)), TG(14:0/22:2(13Z,16Z)/18:1(9Z)), TG(14:0/18:3(9Z,12Z,15Z)/22:0), TG(16:0/18:0/20:3(5Z,8Z,11Z)), TG(16:0/18:0/20:3n6), TG(16:0/20:0/18:3(6Z,9Z,12Z)), TG(16:0/20:0/18:3(9Z,12Z,15Z)), TG(16:0/16:1(9Z)/22:2(13Z,16Z)), TG(16:0/18:1(11Z)/20:2n6), TG(16:0/18:1(9Z)/20:2n6), TG(16:0/20:1(11Z)/18:2(9Z,12Z)), TG(16:0/20:3(5Z,8Z,11Z)/18:0), TG(16:0/18:3(6Z,9Z,12Z)/20:0), TG(16:0/20:2n6/18:1(11Z)), TG(16:0/20:2n6/18:1(9Z)), TG(16:0/20:3n6/18:0), TG(16:0/22:2(13Z,16Z)/16:1(9Z)), TG(16:0/18:3(9Z,12Z,15Z)/20:0), TG(18:0/16:0/20:3(5Z,8Z,11Z)), TG(18:0/16:0/20:3n6),

221

TG(18:0/18:0/18:3(6Z,9Z,12Z)), TG(18:0/18:0/18:3(9Z,12Z,15Z)), TG(18:0/14:1(9Z)/22:2(13Z,16Z)), TG(18:0/16:1(9Z)/20:2n6), TG(18:0/18:1(11Z)/18:2(9Z,12Z)), TG(18:0/18:2(9Z,12Z)/18:1(11Z)), TG(18:0/18:2(9Z,12Z)/18:1(9Z)), TG(18:0/18:3(6Z,9Z,12Z)/18:0)

C51H101O8P M+Na PA(24:0/24:0), PA(i-24:0/i-24:0)

222

TG(16:0/18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), TG(16:1(9Z)/18:0/20:4(5Z,8Z,11Z,14Z)), TG(18:1(9Z)/18:2(9Z,12Z)/18:2(9Z,12Z)), TG(18:1(11Z)/16:0/20:4(5Z,8Z,11Z,14Z)), TG(18:1(9Z)/18:1(9Z)/18:3(6Z,9Z,12Z)), TG(18:1(9Z)/18:1(9Z)/18:3(9Z,12Z,15Z)), TG(18:2(9Z,12Z)/18:0/18:3(9Z,12Z,15Z)), TG(18:2(9Z,12Z)/18:1(11Z)/18:2(9Z,12Z)), Glycerol 1,2-di- (9Z,12Z-octadecadienoate) 3-(9Z-octadecenoate), Glycerol 1,3- di-(9Z,12Z-octadecadienoate) 2-(9Z-octadecenoate), TG(14:0/18:0/22:5(4Z,7Z,10Z,13Z,16Z)), TG(14:0/18:0/22:5(7Z,10Z,13Z,16Z,19Z)), TG(14:0/20:0/20:5(5Z,8Z,11Z,14Z,17Z)), TG(14:0/18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), TG(14:0/18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), TG(14:0/20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), TG(14:0/20:1(11Z)/20:4(8Z,11Z,14Z,17Z)), TG(14:0/20:3(5Z,8Z,11Z)/20:2n6), TG(14:0/22:1(13Z)/18:4(6Z,9Z,12Z,15Z)), TG(14:0/18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), TG(14:0/20:2n6/20:3(5Z,8Z,11Z)), TG(14:0/20:2n6/20:3n6), TG(14:0/20:3n6/20:2n6), TG(14:0/20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), C57H100O6 M+H TG(14:0/22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), TG(14:0/22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), TG(14:0/22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), TG(14:0/22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), TG(14:0/22:5(4Z,7Z,10Z,13Z,16Z)/18:0), TG(14:0/18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), TG(14:0/18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), TG(14:0/20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), TG(14:0/20:5(5Z,8Z,11Z,14Z,17Z)/20:0), TG(14:0/22:5(7Z,10Z,13Z,16Z,19Z)/18:0), TG(16:0/16:0/22:5(4Z,7Z,10Z,13Z,16Z)), TG(16:0/16:0/22:5(7Z,10Z,13Z,16Z,19Z)), TG(16:0/18:0/20:5(5Z,8Z,11Z,14Z,17Z)), TG(16:0/16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), TG(16:0/18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), TG(16:0/18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), TG(16:0/18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), TG(16:0/20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), TG(16:0/20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), TG(16:0/18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), TG(16:0/18:2(9Z,12Z)/20:3n6), TG(16:0/18:3(6Z,9Z,12Z)/20:2n6), TG(16:0/20:2n6/18:3(6Z,9Z,12Z)),

223

TG(16:0/20:2n6/18:3(9Z,12Z,15Z)), TG(16:0/20:3n6/18:2(9Z,12Z)), TG(16:0/20:4(5Z,8Z,11Z,14Z)/18:1(11Z))

224

TG(16:0/18:0/18:2(9Z,12Z)), TG(16:0/16:1(9Z)/20:1(11Z)), TG(16:0/18:1(9Z)/18:1(9Z)), TG(16:1(9Z)/18:0/18:1(9Z)), TG(16:1(9Z)/16:1(9Z)/20:0), TG(16:1(9Z)/18:0/18:1(11Z)), TG(18:1(11Z)/16:0/18:1(11Z)), TG(18:1(11Z)/16:0/18:1(9Z)), Glycerol 2,3-di-(9Z-octadecenoate) 1-hexadecanoate, TG(14:0/16:0/22:2(13Z,16Z)), TG(14:0/18:0/20:2n6), TG(14:0/20:0/18:2(9Z,12Z)), TG(14:0/14:1(9Z)/24:1(15Z)), TG(14:0/16:1(9Z)/22:1(13Z)), TG(14:0/18:1(11Z)/20:1(11Z)), TG(14:0/18:1(9Z)/20:1(11Z)), TG(14:0/20:1(11Z)/18:1(11Z)), TG(14:0/20:1(11Z)/18:1(9Z)), TG(14:0/22:1(13Z)/16:1(9Z)), TG(14:0/24:1(15Z)/14:1(9Z)), TG(14:0/18:2(9Z,12Z)/20:0), C55H102O6 M+Na TG(14:0/20:2n6/18:0), TG(14:0/22:2(13Z,16Z)/16:0), TG(15:0/15:0/22:2(13Z,16Z)), TG(15:0/22:2(13Z,16Z)/15:0), TG(16:0/14:0/22:2(13Z,16Z)), TG(16:0/16:0/20:2n6), TG(16:0/14:1(9Z)/22:1(13Z)), TG(16:0/18:1(9Z)/18:1(11Z)), TG(16:0/18:1(11Z)/18:1(11Z)), TG(16:0/18:1(11Z)/18:1(9Z)), TG(16:0/20:1(11Z)/16:1(9Z)), TG(16:0/22:1(13Z)/14:1(9Z)), TG(16:0/18:2(9Z,12Z)/18:0), TG(16:0/20:2n6/16:0), TG(18:0/14:0/20:2n6), TG(18:0/16:0/18:2(9Z,12Z)), TG(18:0/14:1(9Z)/20:1(11Z)), TG(18:0/16:1(9Z)/18:1(11Z)), TG(18:0/16:1(9Z)/18:1(9Z)), TG(18:0/18:1(11Z)/16:1(9Z)), TG(18:0/18:1(9Z)/16:1(9Z)), TG(18:0/20:1(11Z)/14:1(9Z)), TG(20:0/14:0/18:2(9Z,12Z)), TG(20:0/14:1(9Z)/18:1(11Z)), TG(20:0/14:1(9Z)/18:1(9Z)), TG(20:0/16:1(9Z)/16:1(9Z)), TG(20:0/18:1(11Z)/14:1(9Z)), TG(20:0/18:1(9Z)/14:1(9Z)), TG(22:0/14:1(9Z)/16:1(9Z))

225

PC(20:4(5Z,8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:5(4Z,7Z,10Z,13Z,16Z)), C50H80NO8P M+Na PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe-NMe(22:5(7Z,10Z,13Z,16Z,19Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe-NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/22:4(7Z,10Z,13Z,16Z)), Pe-NMe(22:4(7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z))

PC(20:2(11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:3(5Z,8Z,11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:3(5Z,8Z,11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:3(8Z,11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:3(8Z,11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), C50H84NO8P M+H PC(22:4(7Z,10Z,13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:4(7Z,10Z,13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:3(5Z,8Z,11Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:3(8Z,11Z,14Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:3(5Z,8Z,11Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z)), Pe- NMe(22:2(13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/22:2(13Z,16Z))

226

PC(18:0/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:0/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), PC(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PC(18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), PC(18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), PC(18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), PC(20:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:1(11Z)/20:4(8Z,11Z,14Z,17Z)), PC(20:2(11Z,14Z)/20:3(5Z,8Z,11Z)), PC(20:2(11Z,14Z)/20:3(8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/20:2(11Z,14Z)), PC(20:3(8Z,11Z,14Z)/20:2(11Z,14Z)), PC(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), PC(20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:0), PC(22:1(13Z)/18:4(6Z,9Z,12Z,15Z)), PC(22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), PC(22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), PC(22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), C48H86NO8P M+Na PC(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:0), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/24:1(15Z)), Pe- NMe(20:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(20:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:1(11Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(20:3(5Z,8Z,11Z)/22:2(13Z,16Z)), Pe- NMe(20:3(8Z,11Z,14Z)/22:2(13Z,16Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/22:1(13Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/22:1(13Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/22:0), Pe- NMe(22:0/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(22:1(13Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(22:1(13Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(22:2(13Z,16Z)/20:3(5Z,8Z,11Z)), Pe- NMe(22:2(13Z,16Z)/20:3(8Z,11Z,14Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/20:1(11Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/20:0), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/20:0), Pe- NMe(24:1(15Z)/18:4(6Z,9Z,12Z,15Z))

227

PC(20:3(5Z,8Z,11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), C50H82NO8P M+H PC(22:4(7Z,10Z,13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(5Z,8Z,11Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/22:4(7Z,10Z,13Z,16Z))

228

PC(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PC(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), PC(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), PC(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), PC(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), PC(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PC(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PC(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), PC(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), PC(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PC(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), C48H84NO8P M+Na PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0), Pe- NMe(20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(20:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:2(11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/22:2(13Z,16Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/22:2(13Z,16Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/22:1(13Z)), Pe- NMe(22:1(13Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(22:2(13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(22:2(13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/20:2(11Z,14Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/20:1(11Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/20:1(11Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:0) C46H83O10P M+Na PG(18:0/22:4(7Z,10Z,13Z,16Z))

229

PC(16:0/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/20:3(5Z,8Z,11Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:1(11Z)), PC(18:3(9Z,12Z,15Z)/20:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PC(20:1(11Z)/18:3(6Z,9Z,12Z)), PC(20:1(11Z)/18:3(9Z,12Z,15Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/18:1(9Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(20:4(8Z,11Z,14Z,17Z)/18:0), C46H84NO8P M+K PC(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe(18:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:2(9Z,12Z)/22:2(13Z,16Z)), Pe- NMe(18:3(6Z,9Z,12Z)/22:1(13Z)), Pe- NMe(18:3(9Z,12Z,15Z)/22:1(13Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:0), Pe- NMe(20:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:1(11Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:0), Pe- NMe(22:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:1(13Z)/18:3(6Z,9Z,12Z)), Pe- NMe(22:1(13Z)/18:3(9Z,12Z,15Z)), Pe- NMe(22:2(13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:0)

C45H79NO10P M+Na PC(DiMe(9,3)/MonoMe(11,3)), PC(DiMe(9,3)/MonoMe(9,5)), PC(MonoMe(11,3)/DiMe(9,3)), PC(MonoMe(9,5)/DiMe(9,3))

230

PG(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PG(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PG(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PG(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PG(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PG(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), C46H79O10P M+Na PG(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), PG(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PG(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), PG(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PG(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), PG(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), PG(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PG(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0) C41H82O13P2 M+H PGP(a-13:0/i-22:0), PGP(i-13:0/i-22:0)

231

PC(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:3(6Z,9Z,12Z)/20:3(5Z,8Z,11Z)), PC(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PC(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), PC(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:3(5Z,8Z,11Z)/18:3(6Z,9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:3(9Z,12Z,15Z)), PC(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PC(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(11Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/16:1(9Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), C46H80NO8P M+K PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), Pe- NMe(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), Pe- NMe(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe-

232

NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0)

233

PE(20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(20:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(20:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(20:2(11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), PE(20:4(5Z,8Z,11Z,14Z)/22:2(13Z,16Z)), PE(20:4(8Z,11Z,14Z,17Z)/22:2(13Z,16Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/22:1(13Z)), PE(22:1(13Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(22:2(13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PE(22:2(13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:4(7Z,10Z,13Z,16Z)/20:2(11Z,14Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/20:1(11Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/20:1(11Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:0), Pe- NMe2(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- C47H82NO8P M+Na NMe2(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), Pe- NMe2(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), Pe- NMe2(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0) C47H95N2O6P M+Na SM(d18:0/24:1(15Z))

234

PC(14:1(9Z)/24:1(15Z)), PC(16:0/22:2(13Z,16Z)), PC(16:1(9Z)/22:1(13Z)), PC(18:0/20:2(11Z,14Z)), PC(18:1(11Z)/20:1(11Z)), PC(18:1(9Z)/20:1(11Z)), PC(18:2(9Z,12Z)/20:0), PC(20:0/18:2(9Z,12Z)), C46H88NO8P M+Na PC(20:1(11Z)/18:1(11Z)), PC(20:1(11Z)/18:1(9Z)), PC(20:2(11Z,14Z)/18:0), PC(22:1(13Z)/16:1(9Z)), PC(22:2(13Z,16Z)/16:0), PC(24:1(15Z)/14:1(9Z)), Pe- NMe(16:1(9Z)/24:1(15Z)), Pe-NMe(18:0/22:2(13Z,16Z)), Pe- NMe(18:1(11Z)/22:1(13Z)), Pe-NMe(18:1(9Z)/22:1(13Z)), Pe- NMe(18:2(9Z,12Z)/22:0), Pe-NMe(20:0/20:2(11Z,14Z)), Pe- NMe(20:1(11Z)/20:1(11Z)), Pe-NMe(20:2(11Z,14Z)/20:0), Pe- NMe(22:0/18:2(9Z,12Z)), Pe-NMe(22:1(13Z)/18:1(11Z)), Pe- NMe(22:1(13Z)/18:1(9Z)), Pe-NMe(22:2(13Z,16Z)/18:0), Pe- NMe(24:1(15Z)/16:1(9Z)) C47H93N2O6P M+Na SM(d18:1/24:1(15Z)) C57H86O4 M+H 3-Decaprenyl-4,5-dihydroxybenzoate

235

PC(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PC(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), PC(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), PC(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), PC(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), PC(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PC(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PC(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), PC(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), PC(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PC(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), C48H84NO8P M+H PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0), Pe- NMe(20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(20:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(20:2(11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/22:2(13Z,16Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/22:2(13Z,16Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/22:1(13Z)), Pe- NMe(22:1(13Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(22:2(13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(22:2(13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/20:2(11Z,14Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/20:1(11Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/20:1(11Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:0)

236

PC(16:1(9Z)/22:2(13Z,16Z)), PC(18:0/20:3(5Z,8Z,11Z)), PC(18:0/20:3(8Z,11Z,14Z)), PC(18:1(11Z)/20:2(11Z,14Z)), PC(18:1(9Z)/20:2(11Z,14Z)), PC(18:2(9Z,12Z)/20:1(11Z)), PC(18:3(6Z,9Z,12Z)/20:0), PC(18:3(9Z,12Z,15Z)/20:0), PC(20:0/18:3(6Z,9Z,12Z)), PC(20:0/18:3(9Z,12Z,15Z)), PC(20:1(11Z)/18:2(9Z,12Z)), PC(20:2(11Z,14Z)/18:1(11Z)), PC(20:2(11Z,14Z)/18:1(9Z)), PC(20:3(5Z,8Z,11Z)/18:0), C46H86NO8P M+Na PC(20:3(8Z,11Z,14Z)/18:0), PC(22:2(13Z,16Z)/16:1(9Z)), Pe- NMe(18:1(11Z)/22:2(13Z,16Z)), Pe- NMe(18:1(9Z)/22:2(13Z,16Z)), Pe- NMe(18:2(9Z,12Z)/22:1(13Z)), Pe- NMe(18:3(6Z,9Z,12Z)/22:0), Pe-NMe(18:3(9Z,12Z,15Z)/22:0), Pe-NMe(20:0/20:3(5Z,8Z,11Z)), Pe- NMe(20:0/20:3(8Z,11Z,14Z)), Pe- NMe(20:1(11Z)/20:2(11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:1(11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:0), Pe-NMe(20:3(8Z,11Z,14Z)/20:0), Pe-NMe(22:0/18:3(6Z,9Z,12Z)), Pe- NMe(22:0/18:3(9Z,12Z,15Z)), Pe- NMe(22:1(13Z)/18:2(9Z,12Z)), Pe- NMe(22:2(13Z,16Z)/18:1(11Z)), Pe- NMe(22:2(13Z,16Z)/18:1(9Z))

237

238

PC(16:0/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/20:3(5Z,8Z,11Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:1(11Z)), PC(18:3(9Z,12Z,15Z)/20:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PC(20:1(11Z)/18:3(6Z,9Z,12Z)), PC(20:1(11Z)/18:3(9Z,12Z,15Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/18:1(9Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(20:4(8Z,11Z,14Z,17Z)/18:0), C46H84NO8P M+Na PC(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe(18:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:2(9Z,12Z)/22:2(13Z,16Z)), Pe- NMe(18:3(6Z,9Z,12Z)/22:1(13Z)), Pe- NMe(18:3(9Z,12Z,15Z)/22:1(13Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:0), Pe- NMe(20:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:1(11Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:0), Pe- NMe(22:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:1(13Z)/18:3(6Z,9Z,12Z)), Pe- NMe(22:1(13Z)/18:3(9Z,12Z,15Z)), Pe- NMe(22:2(13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:0)

239

PC(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:3(6Z,9Z,12Z)/20:3(5Z,8Z,11Z)), PC(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PC(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), PC(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:3(5Z,8Z,11Z)/18:3(6Z,9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:3(9Z,12Z,15Z)), PC(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PC(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(11Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/16:1(9Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), C46H80NO8P M+Na PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), Pe- NMe(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), Pe- NMe(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe-

240

NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0)

C42H79NO13 M+Na Lactosylceramide (d18:1/12:0)

PG(18:0/22:5(4Z,7Z,10Z,13Z,16Z)), PG(18:0/22:5(7Z,10Z,13Z,16Z,19Z)), PG(18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), C46H81O10P M+H PG(18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), PG(20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), PG(22:5(4Z,7Z,10Z,13Z,16Z)/18:0), PG(22:5(7Z,10Z,13Z,16Z,19Z)/18:0)

241

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), C44H84NO8P M+K PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine, Pe-NMe(14:1(9Z)/24:1(15Z)), Pe- NMe(16:0/22:2(13Z,16Z)), Pe-NMe(16:1(9Z)/22:1(13Z)), Pe- NMe(18:0/20:2(11Z,14Z)), Pe-NMe(18:1(11Z)/20:1(11Z)), Pe- NMe(18:1(9Z)/20:1(11Z)), Pe-NMe(18:2(9Z,12Z)/20:0), Pe- NMe(20:0/18:2(9Z,12Z)), Pe-NMe(20:1(11Z)/18:1(11Z)), Pe- NMe(20:1(11Z)/18:1(9Z)), Pe-NMe(20:2(11Z,14Z)/18:0), Pe- NMe(22:1(13Z)/16:1(9Z)), Pe-NMe(22:2(13Z,16Z)/16:0), Pe- NMe(24:1(15Z)/14:1(9Z)), Pe-NMe2(15:0/22:2(13Z,16Z)), Pe- NMe2(22:2(13Z,16Z)/15:0)

PC(15:0/22:2(13Z,16Z)), PC(22:2(13Z,16Z)/15:0), PE(16:1(9Z)/24:1(15Z)), PE(18:0/22:2(13Z,16Z)), PE(18:1(11Z)/22:1(13Z)), PE(18:1(9Z)/22:1(13Z)), PE(18:2(9Z,12Z)/22:0), PE(20:0/20:2(11Z,14Z)), C45H86NO8P M+Na PE(20:1(11Z)/20:1(11Z)), PE(20:2(11Z,14Z)/20:0), PE(22:0/18:2(9Z,12Z)), PE(22:1(13Z)/18:1(11Z)), PE(22:1(13Z)/18:1(9Z)), PE(22:2(13Z,16Z)/18:0), PE(24:1(15Z)/16:1(9Z)), Pe-NMe2(14:1(9Z)/24:1(15Z)), Pe- NMe2(16:0/22:2(13Z,16Z)), Pe-NMe2(16:1(9Z)/22:1(13Z)), Pe- NMe2(18:0/20:2(11Z,14Z)), Pe-NMe2(18:1(11Z)/20:1(11Z)), Pe-NMe2(18:1(9Z)/20:1(11Z)), Pe-NMe2(18:2(9Z,12Z)/20:0), Pe-NMe2(20:0/18:2(9Z,12Z)), Pe-NMe2(20:1(11Z)/18:1(11Z)), Pe-NMe2(20:1(11Z)/18:1(9Z)), Pe-NMe2(20:2(11Z,14Z)/18:0), Pe-NMe2(22:1(13Z)/16:1(9Z)), Pe-NMe2(22:2(13Z,16Z)/16:0), Pe-NMe2(24:1(15Z)/14:1(9Z))

242

PG(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PG(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PG(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PG(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), C46H77O10P M+H PG(20:4(5Z,8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PG(20:4(8Z,11Z,14Z,17Z)/20:3(5Z,8Z,11Z)), PG(20:4(8Z,11Z,14Z,17Z)/20:3(8Z,11Z,14Z)), PG(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), PG(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z))

PG(16:0/22:4(7Z,10Z,13Z,16Z)), PG(18:0/20:4(5Z,8Z,11Z,14Z)), PG(18:1(11Z)/20:3(5Z,8Z,11Z)), C44H79O10P M+Na PG(18:1(11Z)/20:3(8Z,11Z,14Z)), PG(18:1(9Z)/20:3(5Z,8Z,11Z)), PG(18:1(9Z)/20:3(8Z,11Z,14Z)), PG(20:2(11Z,14Z)/18:2(9Z,12Z)), PG(20:4(5Z,8Z,11Z,14Z)/18:0)

243

PE(20:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(20:2(11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(20:2(11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(20:3(5Z,8Z,11Z)/22:4(7Z,10Z,13Z,16Z)), PE(20:3(8Z,11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(22:4(7Z,10Z,13Z,16Z)/20:3(5Z,8Z,11Z)), PE(22:4(7Z,10Z,13Z,16Z)/20:3(8Z,11Z,14Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/20:2(11Z,14Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:1(11Z)), Pe- NMe2(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- C47H80NO8P M+H NMe2(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z))

244

PC(15:0/22:4(7Z,10Z,13Z,16Z)), PC(22:4(7Z,10Z,13Z,16Z)/15:0), PE(18:0/22:4(7Z,10Z,13Z,16Z)), PE(18:2(9Z,12Z)/22:2(13Z,16Z)), PE(18:3(6Z,9Z,12Z)/22:1(13Z)), PE(18:3(9Z,12Z,15Z)/22:1(13Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:0), PE(20:0/20:4(5Z,8Z,11Z,14Z)), PE(20:0/20:4(8Z,11Z,14Z,17Z)), PE(20:1(11Z)/20:3(5Z,8Z,11Z)), PE(20:1(11Z)/20:3(8Z,11Z,14Z)), PE(20:2(11Z,14Z)/20:2(11Z,14Z)), PE(20:3(5Z,8Z,11Z)/20:1(11Z)), PE(20:3(8Z,11Z,14Z)/20:1(11Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:0), PE(20:4(8Z,11Z,14Z,17Z)/20:0), PE(22:0/18:4(6Z,9Z,12Z,15Z)), PE(22:1(13Z)/18:3(6Z,9Z,12Z)), PE(22:1(13Z)/18:3(9Z,12Z,15Z)), PE(22:2(13Z,16Z)/18:2(9Z,12Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:0), Pe- NMe2(16:0/22:4(7Z,10Z,13Z,16Z)), Pe- C45H82NO8P M+Na NMe2(18:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:2(9Z,12Z)/20:2(11Z,14Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:1(11Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:1(11Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:0), Pe- NMe2(20:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(20:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:2(11Z,14Z)/18:2(9Z,12Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:1(11Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:1(11Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/16:0)

245

PC(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/15:0), PE(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), PE(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), PE(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), PE(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PE(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), PE(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), C45H78NO8P M+Na PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0), Pe- NMe2(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:2(9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), Pe- NMe2(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(11Z)), Pe-

246

NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/16:1(9Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0)

PC(14:0/22:0), PC(16:0/20:0), PC(18:0/18:0), PC(20:0/16:0), C44H88NO8P M+Na PC(22:0/14:0), PE(15:0/24:0), PE(24:0/15:0), Pe- NMe(14:0/24:0), Pe-NMe(16:0/22:0), Pe-NMe(18:0/20:0), Pe- NMe(20:0/18:0), Pe-NMe(22:0/16:0), Pe-NMe(24:0/14:0), Pe- NMe2(15:0/22:0), Pe-NMe2(22:0/15:0)

247

PC(16:0/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/20:3(5Z,8Z,11Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:1(11Z)), PC(18:3(9Z,12Z,15Z)/20:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PC(20:1(11Z)/18:3(6Z,9Z,12Z)), PC(20:1(11Z)/18:3(9Z,12Z,15Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/18:1(9Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(20:4(8Z,11Z,14Z,17Z)/18:0), C46H84NO8P M+H PC(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe(18:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:2(9Z,12Z)/22:2(13Z,16Z)), Pe- NMe(18:3(6Z,9Z,12Z)/22:1(13Z)), Pe- NMe(18:3(9Z,12Z,15Z)/22:1(13Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:0), Pe- NMe(20:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:1(11Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:0), Pe- NMe(22:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:1(13Z)/18:3(6Z,9Z,12Z)), Pe- NMe(22:1(13Z)/18:3(9Z,12Z,15Z)), Pe- NMe(22:2(13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:0)

248

PC(14:0/22:1(13Z)), PC(14:1(9Z)/22:0), PC(16:0/20:1(11Z)), PC(16:1(9Z)/20:0), PC(18:0/18:1(11Z)), PC(18:0/18:1(9Z)), PC(18:1(11Z)/18:0), PC(18:1(9Z)/18:0), PC(20:0/16:1(9Z)), PC(20:1(11Z)/16:0), PC(22:0/14:1(9Z)), PC(22:1(13Z)/14:0), C44H86NO8P M+Na PE(15:0/24:1(15Z)), PE(24:1(15Z)/15:0), Pe- NMe(14:0/24:1(15Z)), Pe-NMe(14:1(9Z)/24:0), Pe- NMe(16:0/22:1(13Z)), Pe-NMe(16:1(9Z)/22:0), Pe- NMe(18:0/20:1(11Z)), Pe-NMe(18:1(11Z)/20:0), Pe- NMe(18:1(9Z)/20:0), Pe-NMe(20:0/18:1(11Z)), Pe- NMe(20:0/18:1(9Z)), Pe-NMe(20:1(11Z)/18:0), Pe- NMe(22:0/16:1(9Z)), Pe-NMe(22:1(13Z)/16:0), Pe- NMe(24:0/14:1(9Z)), Pe-NMe(24:1(15Z)/14:0), Pe- NMe2(15:0/22:1(13Z)), Pe-NMe2(22:1(13Z)/15:0)

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), C44H84NO8P M+Na PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine, Pe-NMe(14:1(9Z)/24:1(15Z)), Pe- NMe(16:0/22:2(13Z,16Z)), Pe-NMe(16:1(9Z)/22:1(13Z)), Pe- NMe(18:0/20:2(11Z,14Z)), Pe-NMe(18:1(11Z)/20:1(11Z)), Pe- NMe(18:1(9Z)/20:1(11Z)), Pe-NMe(18:2(9Z,12Z)/20:0), Pe- NMe(20:0/18:2(9Z,12Z)), Pe-NMe(20:1(11Z)/18:1(11Z)), Pe- NMe(20:1(11Z)/18:1(9Z)), Pe-NMe(20:2(11Z,14Z)/18:0), Pe- NMe(22:1(13Z)/16:1(9Z)), Pe-NMe(22:2(13Z,16Z)/16:0), Pe- NMe(24:1(15Z)/14:1(9Z)), Pe-NMe2(15:0/22:2(13Z,16Z)), Pe- NMe2(22:2(13Z,16Z)/15:0)

249

PC(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(18:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:3(6Z,9Z,12Z)/20:3(5Z,8Z,11Z)), PC(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PC(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), PC(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:3(5Z,8Z,11Z)/18:3(6Z,9Z,12Z)), PC(20:3(5Z,8Z,11Z)/18:3(9Z,12Z,15Z)), PC(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PC(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(11Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/16:1(9Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), C46H80NO8P M+H PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/22:2(13Z,16Z)), Pe- NMe(20:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(20:2(11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:2(11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/20:1(11Z)), Pe- NMe(22:2(13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(11Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:1(9Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe-

250

NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0)

251

PC(14:1(9Z)/22:2(13Z,16Z)), PC(16:0/20:3(5Z,8Z,11Z)), PC(16:0/20:3(8Z,11Z,14Z)), PC(16:1(9Z)/20:2(11Z,14Z)), PC(18:0/18:3(6Z,9Z,12Z)), PC(18:0/18:3(9Z,12Z,15Z)), PC(18:1(11Z)/18:2(9Z,12Z)), PC(18:1(9Z)/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:1(9Z)), PC(18:3(6Z,9Z,12Z)/18:0), PC(18:3(9Z,12Z,15Z)/18:0), PC(20:2(11Z,14Z)/16:1(9Z)), PC(20:3(5Z,8Z,11Z)/16:0), C44H82NO8P M+Na PC(20:3(8Z,11Z,14Z)/16:0), PC(22:2(13Z,16Z)/14:1(9Z)), Pe- NMe(16:1(9Z)/22:2(13Z,16Z)), Pe- NMe(18:0/20:3(5Z,8Z,11Z)), Pe-NMe(18:0/20:3(8Z,11Z,14Z)), Pe-NMe(18:1(11Z)/20:2(11Z,14Z)), Pe- NMe(18:1(9Z)/20:2(11Z,14Z)), Pe- NMe(18:2(9Z,12Z)/20:1(11Z)), Pe- NMe(18:3(6Z,9Z,12Z)/20:0), Pe-NMe(18:3(9Z,12Z,15Z)/20:0), Pe-NMe(20:0/18:3(6Z,9Z,12Z)), Pe- NMe(20:0/18:3(9Z,12Z,15Z)), Pe- NMe(20:1(11Z)/18:2(9Z,12Z)), Pe- NMe(20:2(11Z,14Z)/18:1(11Z)), Pe- NMe(20:2(11Z,14Z)/18:1(9Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:0), Pe-NMe(20:3(8Z,11Z,14Z)/18:0), Pe-NMe(22:2(13Z,16Z)/16:1(9Z)) C42H79NO13 M+H Lactosylceramide (d18:1/12:0)

252

PC(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(18:3(6Z,9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:3(9Z,12Z,15Z)/20:4(5Z,8Z,11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:3(8Z,11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:3(6Z,9Z,12Z)), PC(20:4(8Z,11Z,14Z,17Z)/18:3(9Z,12Z,15Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), Pe- NMe(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- C46H78NO8P M+H NMe(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:3(5Z,8Z,11Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(20:3(8Z,11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:3(5Z,8Z,11Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/20:3(8Z,11Z,14Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z))

253

PC(14:0/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(16:0/20:4(8Z,11Z,14Z,17Z)), PC(16:1(9Z)/20:3(5Z,8Z,11Z)), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PC(18:1(11Z)/18:3(6Z,9Z,12Z)), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(18:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(20:3(5Z,8Z,11Z)/16:1(9Z)), PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PC(20:4(8Z,11Z,14Z,17Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe(16:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:0/20:4(5Z,8Z,11Z,14Z)), Pe- C44H80NO8P M+Na NMe(18:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(18:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(18:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe(18:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:2(9Z,12Z)/20:2(11Z,14Z)), Pe- NMe(18:3(6Z,9Z,12Z)/20:1(11Z)), Pe- NMe(18:3(9Z,12Z,15Z)/20:1(11Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/20:0), Pe- NMe(20:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(20:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe(20:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe(20:2(11Z,14Z)/18:2(9Z,12Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:1(11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:1(9Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:1(9Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/18:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/18:0), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe2(15:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/15:0)

254

PG(16:0/22:4(7Z,10Z,13Z,16Z)), PG(18:0/20:4(5Z,8Z,11Z,14Z)), PG(18:1(11Z)/20:3(5Z,8Z,11Z)), C44H79O10P M+H PG(18:1(11Z)/20:3(8Z,11Z,14Z)), PG(18:1(9Z)/20:3(5Z,8Z,11Z)), PG(18:1(9Z)/20:3(8Z,11Z,14Z)), PG(20:2(11Z,14Z)/18:2(9Z,12Z)), PG(20:4(5Z,8Z,11Z,14Z)/18:0) C42H81O10P M+Na PG(18:0/18:1(11Z)), PG(18:0/18:1(9Z)), PG(18:1(11Z)/18:0), PG(18:1(9Z)/18:0)

PE(22:5(4Z,7Z,10Z,13Z,16Z)/P-18:1(11Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/P-18:1(9Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/P-18:1(11Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/P-18:1(9Z)), C45H78NO7P M+Na PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/P-18:0), PE(P- 18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(P- 18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(P- 18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(P- 18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(P- 18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)) C41H78NO10P M+Na PS(15:0/20:1(11Z)), PS(20:1(11Z)/15:0)

255

PG(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), PG(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), PG(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PG(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PG(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), C44H77O10P M+H PG(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), PG(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), PG(20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), PG(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), PG(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PG(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), PG(22:5(7Z,10Z,13Z,16Z,19Z)/16:0), PG(22:5(4Z,7Z,10Z,13Z,16Z)/16:0) PG(18:0/18:2(9Z,12Z)), PG(18:1(11Z)/18:1(11Z)), C42H79O10P M+Na PG(18:1(11Z)/18:1(9Z)), PG(18:1(9Z)/18:1(11Z)), PG(18:1(9Z)/18:1(9Z)), PG(18:2(9Z,12Z)/18:0)

PC(15:0/20:1(11Z)), PC(20:1(11Z)/15:0), PE(14:0/24:1(15Z)), PE(14:1(9Z)/24:0), PE(16:0/22:1(13Z)), PE(16:1(9Z)/22:0), PE(18:0/20:1(11Z)), PE(18:1(11Z)/20:0), PE(18:1(9Z)/20:0), PE(20:0/18:1(11Z)), PE(20:0/18:1(9Z)), PE(20:1(11Z)/18:0), PE(22:1(13Z)/16:0), PE(22:0/16:1(9Z)), PE(24:0/14:1(9Z)), C43H84NO8P M+Na PE(24:1(15Z)/14:0), Pe-NMe(15:0/22:1(13Z)), Pe- NMe(22:1(13Z)/15:0), Pe-NMe2(18:0/18:1(9Z)), Pe- NMe2(18:0/18:1(11Z)), Pe-NMe2(14:1(9Z)/22:0), Pe- NMe2(16:0/20:1(11Z)), Pe-NMe2(16:1(9Z)/20:0), Pe- NMe2(14:0/22:1(13Z)), Pe-NMe2(18:1(11Z)/18:0), Pe- NMe2(18:1(9Z)/18:0), Pe-NMe2(20:0/16:1(9Z)), Pe- NMe2(20:1(11Z)/16:0), Pe-NMe2(22:0/14:1(9Z)), Pe- NMe2(22:1(13Z)/14:0) PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/P-18:1(11Z)), C45H76NO7P M+Na PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/P-18:1(9Z)), PE(P- 18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(P- 18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z))

256

257

NMe2(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:1(11Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:1(9Z))

258

PC(15:0/20:2(11Z,14Z)), PC(20:2(11Z,14Z)/15:0), PE(14:1(9Z)/24:1(15Z)), PE(16:0/22:2(13Z,16Z)), PE(16:1(9Z)/22:1(13Z)), PE(18:0/20:2(11Z,14Z)), PE(18:1(11Z)/20:1(11Z)), PE(18:1(9Z)/20:1(11Z)), C43H82NO8P M+Na PE(18:2(9Z,12Z)/20:0), PE(20:0/18:2(9Z,12Z)), PE(20:1(11Z)/18:1(11Z)), PE(20:1(11Z)/18:1(9Z)), PE(20:2(11Z,14Z)/18:0), PE(22:1(13Z)/16:1(9Z)), PE(22:2(13Z,16Z)/16:0), PE(24:1(15Z)/14:1(9Z)), Pe- NMe2(18:1(9Z)/18:1(9Z)), Pe-NMe(15:0/22:2(13Z,16Z)), Pe- NMe(22:2(13Z,16Z)/15:0), Pe-NMe2(16:1(9Z)/20:1(11Z)), Pe- NMe2(18:1(9Z)/18:1(11Z)), Pe-NMe2(18:1(11Z)/18:1(9Z)), Pe- NMe2(18:1(11Z)/18:1(11Z)), Pe-NMe2(14:0/22:2(13Z,16Z)), Pe-NMe2(14:1(9Z)/22:1(13Z)), Pe-NMe2(16:0/20:2(11Z,14Z)), Pe-NMe2(18:0/18:2(9Z,12Z)), Pe-NMe2(18:2(9Z,12Z)/18:0), Pe-NMe2(20:1(11Z)/16:1(9Z)), Pe-NMe2(20:2(11Z,14Z)/16:0), Pe-NMe2(22:1(13Z)/14:1(9Z)), Pe-NMe2(22:2(13Z,16Z)/14:0)

259

PC(15:0/20:3(5Z,8Z,11Z)), PC(15:0/20:3(8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/15:0), PC(20:3(8Z,11Z,14Z)/15:0), PE(16:1(9Z)/22:2(13Z,16Z)), PE(18:0/20:3(5Z,8Z,11Z)), PE(18:0/20:3(8Z,11Z,14Z)), PE(18:1(11Z)/20:2(11Z,14Z)), PE(18:1(9Z)/20:2(11Z,14Z)), PE(18:2(9Z,12Z)/20:1(11Z)), PE(18:3(6Z,9Z,12Z)/20:0), PE(18:3(9Z,12Z,15Z)/20:0), PE(20:0/18:3(6Z,9Z,12Z)), PE(20:0/18:3(9Z,12Z,15Z)), PE(20:1(11Z)/18:2(9Z,12Z)), PE(20:2(11Z,14Z)/18:1(11Z)), PE(20:2(11Z,14Z)/18:1(9Z)), PE(20:3(5Z,8Z,11Z)/18:0), PE(20:3(8Z,11Z,14Z)/18:0), PE(22:2(13Z,16Z)/16:1(9Z)), Pe- NMe2(14:1(9Z)/22:2(13Z,16Z)), Pe- C43H80NO8P M+Na NMe2(16:0/20:3(5Z,8Z,11Z)), Pe- NMe2(16:0/20:3(8Z,11Z,14Z)), Pe- NMe2(16:1(9Z)/20:2(11Z,14Z)), Pe- NMe2(18:0/18:3(6Z,9Z,12Z)), Pe- NMe2(18:0/18:3(9Z,12Z,15Z)), Pe- NMe2(18:1(11Z)/18:2(9Z,12Z)), Pe- NMe2(18:1(9Z)/18:2(9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/18:1(11Z)), Pe- NMe2(18:2(9Z,12Z)/18:1(9Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:0), Pe- NMe2(18:3(9Z,12Z,15Z)/18:0), Pe- NMe2(20:2(11Z,14Z)/16:1(9Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/16:0), Pe- NMe2(20:3(8Z,11Z,14Z)/16:0), Pe- NMe2(22:2(13Z,16Z)/14:1(9Z))

260

PE(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), PE(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:3(5Z,8Z,11Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:3(5Z,8Z,11Z)/20:4(8Z,11Z,14Z,17Z)), PE(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/20:4(8Z,11Z,14Z,17Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:3(5Z,8Z,11Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:3(5Z,8Z,11Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:3(8Z,11Z,14Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,12Z)), C45H76NO8P M+H PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(11Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), Pe- NMe2(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(20:3(5Z,8Z,11Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:1(9Z))

261

PC(15:0/20:4(5Z,8Z,11Z,14Z)), PC(15:0/20:4(8Z,11Z,14Z,17Z)), PC(20:4(5Z,8Z,11Z,14Z)/15:0), PC(20:4(8Z,11Z,14Z,17Z)/15:0), PE(16:0/22:4(7Z,10Z,13Z,16Z)), PE(16:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:0/20:4(5Z,8Z,11Z,14Z)), PE(18:0/20:4(8Z,11Z,14Z,17Z)), PE(18:1(11Z)/20:3(5Z,8Z,11Z)), PE(18:1(11Z)/20:3(8Z,11Z,14Z)), PE(18:1(9Z)/20:3(5Z,8Z,11Z)), PE(18:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:2(9Z,12Z)/20:2(11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:1(11Z)), PE(18:3(9Z,12Z,15Z)/20:1(11Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:0), PE(20:0/18:4(6Z,9Z,12Z,15Z)), PE(20:1(11Z)/18:3(6Z,9Z,12Z)), PE(20:1(11Z)/18:3(9Z,12Z,15Z)), PE(20:2(11Z,14Z)/18:2(9Z,12Z)), PE(20:3(5Z,8Z,11Z)/18:1(11Z)), PE(20:3(5Z,8Z,11Z)/18:1(9Z)), PE(20:3(8Z,11Z,14Z)/18:1(11Z)), PE(20:3(8Z,11Z,14Z)/18:1(9Z)), C43H78NO8P M+Na PE(20:4(5Z,8Z,11Z,14Z)/18:0), PE(20:4(8Z,11Z,14Z,17Z)/18:0), PE(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe(15:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/15:0), Pe- NMe2(14:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(16:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(16:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(16:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:2(9Z,12Z)/18:2(9Z,12Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:1(11Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:1(9Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:1(11Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:1(9Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:0), Pe- NMe2(20:3(5Z,8Z,11Z)/16:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/16:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/16:0), Pe-

262

NMe2(20:4(8Z,11Z,14Z,17Z)/16:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe2(16:0/20:4(8Z,11Z,14Z,17Z))

263

PC(15:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/15:0), PE(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), PE(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), PE(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:0/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), PE(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:2(11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:2(11Z,14Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:1(11Z)), PE(20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:2(11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:2(11Z,14Z)/18:3(9Z,12Z,15Z)), PE(20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), PE(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:1(11Z)), C43H76NO8P M+Na PE(20:4(8Z,11Z,14Z,17Z)/18:1(9Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:0), PE(22:4(7Z,10Z,13Z,16Z)/16:1(9Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/16:0), PE(22:5(7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(15:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(15:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/15:0), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/15:0), Pe- NMe2(14:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(14:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(14:1(9Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(16:0/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(16:1(9Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(16:1(9Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(18:1(11Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:1(9Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:2(9Z,12Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:2(9Z,12Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:2(9Z,12Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:1(11Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:1(9Z)), Pe-

264

NMe2(20:4(5Z,8Z,11Z,14Z)/16:1(9Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/16:1(9Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/16:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/14:1(9Z))

265

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), C44H84NO8P M+H PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine, Pe-NMe(14:1(9Z)/24:1(15Z)), Pe- NMe(16:0/22:2(13Z,16Z)), Pe-NMe(16:1(9Z)/22:1(13Z)), Pe- NMe(18:0/20:2(11Z,14Z)), Pe-NMe(18:1(11Z)/20:1(11Z)), Pe- NMe(18:1(9Z)/20:1(11Z)), Pe-NMe(18:2(9Z,12Z)/20:0), Pe- NMe(20:0/18:2(9Z,12Z)), Pe-NMe(20:1(11Z)/18:1(11Z)), Pe- NMe(20:1(11Z)/18:1(9Z)), Pe-NMe(20:2(11Z,14Z)/18:0), Pe- NMe(22:1(13Z)/16:1(9Z)), Pe-NMe(22:2(13Z,16Z)/16:0), Pe- NMe(24:1(15Z)/14:1(9Z)), Pe-NMe2(15:0/22:2(13Z,16Z)), Pe- NMe2(22:2(13Z,16Z)/15:0)

PE(18:3(6Z,9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:3(9Z,12Z,15Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:4(8Z,11Z,14Z,17Z)/20:5(5Z,8Z,11Z,14Z,17Z)), C45H72NO8P M+H PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:3(6Z,9Z,12Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/18:4(6Z,9Z,12Z,15Z))

266

PE(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(18:1(11Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:2(9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:3(6Z,9Z,12Z)/20:3(5Z,8Z,11Z)), PE(18:3(6Z,9Z,12Z)/20:3(8Z,11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PE(18:3(9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:3(5Z,8Z,11Z)/18:3(6Z,9Z,12Z)), PE(20:3(5Z,8Z,11Z)/18:3(9Z,12Z,15Z)), PE(20:3(8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:3(8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:2(9Z,12Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(11Z)), C43H74NO8P M+Na PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:1(9Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/16:1(9Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/15:0), Pe- NMe2(14:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(14:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(14:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(16:1(9Z)/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe2(18:2(9Z,12Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:2(9Z,12Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/16:1(9Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/14:1(9Z)), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/14:1(9Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/14:0)

PC(14:0/20:0), PC(16:0/18:0), PC(18:0/16:0), PC(20:0/14:0), C42H84NO8P M+Na PE(15:0/22:0), PE(22:0/15:0), Pe-NMe(18:0/18:0), Pe- NMe(14:0/22:0), Pe-NMe(16:0/20:0), Pe-NMe(20:0/16:0), Pe- NMe(22:0/14:0), Pe-NMe2(15:0/20:0), Pe-NMe2(20:0/15:0)

267

PC(14:0/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(16:0/20:4(8Z,11Z,14Z,17Z)), PC(16:1(9Z)/20:3(5Z,8Z,11Z)), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PC(18:1(11Z)/18:3(6Z,9Z,12Z)), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(18:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(20:3(5Z,8Z,11Z)/16:1(9Z)), PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PC(20:4(8Z,11Z,14Z,17Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe(16:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:0/20:4(5Z,8Z,11Z,14Z)), Pe- C44H80NO8P M+H NMe(18:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(18:1(11Z)/20:3(5Z,8Z,11Z)), Pe- NMe(18:1(11Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe(18:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:2(9Z,12Z)/20:2(11Z,14Z)), Pe- NMe(18:3(6Z,9Z,12Z)/20:1(11Z)), Pe- NMe(18:3(9Z,12Z,15Z)/20:1(11Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/20:0), Pe- NMe(20:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(20:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe(20:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe(20:2(11Z,14Z)/18:2(9Z,12Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:1(11Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:1(9Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:1(11Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:1(9Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/18:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/18:0), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe2(15:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/15:0)

268

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(16:0/18:1(11Z)), PC(16:0/18:1(9Z)), PC(16:1(9Z)/18:0), PC(18:0/16:1(9Z)), PC(18:1(11Z)/16:0), PC(18:1(9Z)/16:0), PC(20:0/14:1(9Z)), PC(20:1(11Z)/14:0), PE(15:0/22:1(13Z)), PE(22:1(13Z)/15:0), C42H82NO8P M+Na Pe-NMe(18:0/18:1(9Z)), Pe-NMe(18:0/18:1(11Z)), Pe- NMe(14:1(9Z)/22:0), Pe-NMe(16:0/20:1(11Z)), Pe- NMe(16:1(9Z)/20:0), Pe-NMe(14:0/22:1(13Z)), Pe- NMe(18:1(11Z)/18:0), Pe-NMe(18:1(9Z)/18:0), Pe- NMe(20:0/16:1(9Z)), Pe-NMe(20:1(11Z)/16:0), Pe- NMe(22:0/14:1(9Z)), Pe-NMe(22:1(13Z)/14:0), Pe- NMe2(15:0/20:1(11Z)), Pe-NMe2(20:1(11Z)/15:0) PC(14:0/22:5(4Z,7Z,10Z,13Z,16Z)), PC(14:0/22:5(7Z,10Z,13Z,16Z,19Z)), PC(14:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(16:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PC(16:1(9Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PC(18:1(9Z)/18:4(6Z,9Z,12Z,15Z)), PC(18:2(9Z,12Z)/18:3(6Z,9Z,12Z)), PC(18:2(9Z,12Z)/18:3(9Z,12Z,15Z)), PC(18:3(6Z,9Z,12Z)/18:2(9Z,12Z)), PC(18:3(9Z,12Z,15Z)/18:2(9Z,12Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:1(9Z)), PC(20:4(8Z,11Z,14Z,17Z)/16:1(9Z)), C44H78NO8P M+H PC(20:5(5Z,8Z,11Z,14Z,17Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/14:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/14:0), Pe- NMe(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(18:0/20:5(5Z,8Z,11Z,14Z,17Z)), Pe- NMe(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), Pe- NMe(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:3(6Z,9Z,12Z)/20:2(11Z,14Z)), Pe- NMe(18:3(9Z,12Z,15Z)/20:2(11Z,14Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/20:1(11Z)), Pe-

269

NMe(20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe(20:2(11Z,14Z)/18:3(6Z,9Z,12Z)), Pe- NMe(20:2(11Z,14Z)/18:3(9Z,12Z,15Z)), Pe- NMe(20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), Pe- NMe(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/18:1(11Z)), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/18:1(9Z)), Pe- NMe(20:5(5Z,8Z,11Z,14Z,17Z)/18:0), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/16:1(9Z)), Pe- NMe(22:5(7Z,10Z,13Z,16Z,19Z)/16:0), Pe- NMe2(15:0/22:5(4Z,7Z,10Z,13Z,16Z)), Pe- NMe2(15:0/22:5(7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(22:5(4Z,7Z,10Z,13Z,16Z)/15:0), Pe- NMe2(22:5(7Z,10Z,13Z,16Z,19Z)/15:0), Pe- NMe(22:5(4Z,7Z,10Z,13Z,16Z)/16:0)

270

PC(14:0/20:2(11Z,14Z)), PC(14:1(9Z)/20:1(11Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(16:1(9Z)/18:1(9Z)), PC(18:1(11Z)/16:1(9Z)), PC(18:1(9Z)/16:1(9Z)), PC(18:2(9Z,12Z)/16:0), C42H80NO8P M+Na PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PE(15:0/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/15:0), Pe- NMe(18:1(9Z)/18:1(9Z)), Pe-NMe(16:1(9Z)/20:1(11Z)), Pe- NMe(18:1(9Z)/18:1(11Z)), Pe-NMe(18:1(11Z)/18:1(9Z)), Pe- NMe(18:1(11Z)/18:1(11Z)), Pe-NMe(14:0/22:2(13Z,16Z)), Pe- NMe(14:1(9Z)/22:1(13Z)), Pe-NMe(16:0/20:2(11Z,14Z)), Pe- NMe(18:0/18:2(9Z,12Z)), Pe-NMe(18:2(9Z,12Z)/18:0), Pe- NMe(20:1(11Z)/16:1(9Z)), Pe-NMe(20:2(11Z,14Z)/16:0), Pe- NMe(22:1(13Z)/14:1(9Z)), Pe-NMe(22:2(13Z,16Z)/14:0), Pe- NMe2(15:0/20:2(11Z,14Z)), Pe-NMe2(20:2(11Z,14Z)/15:0)

271

PC(14:0/20:4(5Z,8Z,11Z,14Z)), PC(14:0/20:4(8Z,11Z,14Z,17Z)), PC(14:1(9Z)/20:3(5Z,8Z,11Z)), PC(14:1(9Z)/20:3(8Z,11Z,14Z)), PC(16:0/18:4(6Z,9Z,12Z,15Z)), PC(16:1(9Z)/18:3(6Z,9Z,12Z)), PC(16:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:3(6Z,9Z,12Z)/16:1(9Z)), PC(18:3(9Z,12Z,15Z)/16:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/16:0), PC(20:3(5Z,8Z,11Z)/14:1(9Z)), PC(20:3(8Z,11Z,14Z)/14:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/14:0), PC(20:4(8Z,11Z,14Z,17Z)/14:0), PE(15:0/22:4(7Z,10Z,13Z,16Z)), PE(22:4(7Z,10Z,13Z,16Z)/15:0), Pe- NMe(14:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(16:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(16:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe(16:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:0/18:4(6Z,9Z,12Z,15Z)), Pe- C42H76NO8P M+Na NMe(18:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe(18:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe(18:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe(18:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe(18:2(9Z,12Z)/18:2(9Z,12Z)), Pe- NMe(18:3(6Z,9Z,12Z)/18:1(11Z)), Pe- NMe(18:3(6Z,9Z,12Z)/18:1(9Z)), Pe- NMe(18:3(9Z,12Z,15Z)/18:1(11Z)), Pe- NMe(18:3(9Z,12Z,15Z)/18:1(9Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/18:0), Pe- NMe(20:3(5Z,8Z,11Z)/16:1(9Z)), Pe- NMe(20:3(8Z,11Z,14Z)/16:1(9Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/16:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/16:0), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe2(15:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(15:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/15:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/15:0), Pe- NMe(16:0/20:4(8Z,11Z,14Z,17Z)) C46H70O8 M+Na 1,26-Hexacosanediol diferulate, 1,28-Dicaffeoyloctacosanediol

272

PC(15:0/20:4(5Z,8Z,11Z,14Z)), PC(15:0/20:4(8Z,11Z,14Z,17Z)), PC(20:4(5Z,8Z,11Z,14Z)/15:0), PC(20:4(8Z,11Z,14Z,17Z)/15:0), PE(16:0/22:4(7Z,10Z,13Z,16Z)), PE(16:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:0/20:4(5Z,8Z,11Z,14Z)), PE(18:0/20:4(8Z,11Z,14Z,17Z)), PE(18:1(11Z)/20:3(5Z,8Z,11Z)), PE(18:1(11Z)/20:3(8Z,11Z,14Z)), PE(18:1(9Z)/20:3(5Z,8Z,11Z)), PE(18:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:2(9Z,12Z)/20:2(11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:1(11Z)), PE(18:3(9Z,12Z,15Z)/20:1(11Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:0), PE(20:0/18:4(6Z,9Z,12Z,15Z)), PE(20:1(11Z)/18:3(6Z,9Z,12Z)), PE(20:1(11Z)/18:3(9Z,12Z,15Z)), PE(20:2(11Z,14Z)/18:2(9Z,12Z)), PE(20:3(5Z,8Z,11Z)/18:1(11Z)), PE(20:3(5Z,8Z,11Z)/18:1(9Z)), PE(20:3(8Z,11Z,14Z)/18:1(11Z)), PE(20:3(8Z,11Z,14Z)/18:1(9Z)), C43H78NO8P M+H PE(20:4(5Z,8Z,11Z,14Z)/18:0), PE(20:4(8Z,11Z,14Z,17Z)/18:0), PE(22:4(7Z,10Z,13Z,16Z)/16:0), Pe- NMe(15:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/15:0), Pe- NMe2(14:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe2(16:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(16:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(16:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(18:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:2(9Z,12Z)/18:2(9Z,12Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:1(11Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:1(9Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:1(11Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:1(9Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:0), Pe- NMe2(20:3(5Z,8Z,11Z)/16:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/16:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/16:0), Pe-

273

NMe2(20:4(8Z,11Z,14Z,17Z)/16:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe2(16:0/20:4(8Z,11Z,14Z,17Z))

PC(15:0/18:1(11Z)), PC(15:0/18:1(9Z)), PC(18:1(11Z)/15:0), PC(18:1(9Z)/15:0), PE(14:0/22:1(13Z)), PE(14:1(9Z)/22:0), PE(16:0/20:1(11Z)), PE(16:1(9Z)/20:0), PE(18:0/18:1(11Z)), PE(18:0/18:1(9Z)), PE(18:1(11Z)/18:0), PE(18:1(9Z)/18:0), PE(20:0/16:1(9Z)), PE(20:1(11Z)/16:0), PE(22:0/14:1(9Z)), C41H80NO8P M+Na PE(22:1(13Z)/14:0), Pe-NMe2(16:0/18:1(9Z)), Pe- NMe(15:0/20:1(11Z)), Pe-NMe(20:1(11Z)/15:0), Pe- NMe2(14:0/20:1(11Z)), Pe-NMe2(14:1(9Z)/20:0), Pe- NMe2(16:0/18:1(11Z)), Pe-NMe2(16:1(9Z)/18:0), Pe- NMe2(18:0/16:1(9Z)), Pe-NMe2(18:1(11Z)/16:0), Pe- NMe2(18:1(9Z)/16:0), Pe-NMe2(20:0/14:1(9Z)), Pe- NMe2(20:1(11Z)/14:0)

274

275

NMe2(20:4(5Z,8Z,11Z,14Z)/16:1(9Z)), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/16:1(9Z)), Pe- NMe2(20:5(5Z,8Z,11Z,14Z,17Z)/16:0), Pe- NMe2(22:4(7Z,10Z,13Z,16Z)/14:1(9Z))

276

PC(15:0/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/15:0), PE(14:0/22:2(13Z,16Z)), PE(14:1(9Z)/22:1(13Z)), PE(16:0/20:2(11Z,14Z)), PE(16:1(9Z)/20:1(11Z)), PE(18:0/18:2(9Z,12Z)), PE(18:1(11Z)/18:1(11Z)), C41H78NO8P M+Na PE(18:1(11Z)/18:1(9Z)), PE(18:1(9Z)/18:1(11Z)), PE(18:1(9Z)/18:1(9Z)), PE(18:2(9Z,12Z)/18:0), PE(20:1(11Z)/16:1(9Z)), PE(20:2(11Z,14Z)/16:0), PE(22:1(13Z)/14:1(9Z)), PE(22:2(13Z,16Z)/14:0), Pe- NMe(15:0/20:2(11Z,14Z)), Pe-NMe(20:2(11Z,14Z)/15:0), Pe- NMe2(14:1(9Z)/20:1(11Z)), Pe-NMe2(16:1(9Z)/18:1(9Z)), Pe- NMe2(16:1(9Z)/18:1(11Z)), Pe-NMe2(14:0/20:2(11Z,14Z)), Pe- NMe2(16:0/18:2(9Z,12Z)), Pe-NMe2(18:1(9Z)/16:1(9Z)), Pe- NMe2(18:1(11Z)/16:1(9Z)), Pe-NMe2(18:2(9Z,12Z)/16:0), Pe- NMe2(20:1(11Z)/14:1(9Z)), Pe-NMe2(20:2(11Z,14Z)/14:0)

PE(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:3(6Z,9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:3(9Z,12Z,15Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PE(20:3(5Z,8Z,11Z)/18:4(6Z,9Z,12Z,15Z)), C43H72NO8P M+H PE(20:3(8Z,11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:3(6Z,9Z,12Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:3(9Z,12Z,15Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:1(9Z)), Pe- NMe2(14:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/14:1(9Z))

277

PC(15:0/18:4(6Z,9Z,12Z,15Z)), PC(18:4(6Z,9Z,12Z,15Z)/15:0), PE(14:0/22:4(7Z,10Z,13Z,16Z)), PE(16:0/20:4(5Z,8Z,11Z,14Z)), PE(16:0/20:4(8Z,11Z,14Z,17Z)), PE(16:1(9Z)/20:3(5Z,8Z,11Z)), PE(18:0/18:4(6Z,9Z,12Z,15Z)), PE(18:1(11Z)/18:3(6Z,9Z,12Z)), PE(18:1(11Z)/18:3(9Z,12Z,15Z)), PE(18:1(9Z)/18:3(6Z,9Z,12Z)), PE(18:1(9Z)/18:3(9Z,12Z,15Z)), PE(18:2(9Z,12Z)/18:2(9Z,12Z)), PE(18:3(6Z,9Z,12Z)/18:1(11Z)), PE(18:3(6Z,9Z,12Z)/18:1(9Z)), PE(18:3(9Z,12Z,15Z)/18:1(11Z)), PE(18:3(9Z,12Z,15Z)/18:1(9Z)), PE(18:4(6Z,9Z,12Z,15Z)/18:0), PE(20:3(5Z,8Z,11Z)/16:1(9Z)), PE(20:3(8Z,11Z,14Z)/16:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/16:0), PE(20:4(8Z,11Z,14Z,17Z)/16:0), C41H74NO8P M+Na PE(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe(15:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(15:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/15:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/15:0), Pe- NMe2(14:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(14:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(14:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe2(14:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe2(16:0/18:4(6Z,9Z,12Z,15Z)), Pe- NMe2(16:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe2(16:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe2(18:3(6Z,9Z,12Z)/16:1(9Z)), Pe- NMe2(18:3(9Z,12Z,15Z)/16:1(9Z)), Pe- NMe2(18:4(6Z,9Z,12Z,15Z)/16:0), Pe- NMe2(20:3(5Z,8Z,11Z)/14:1(9Z)), Pe- NMe2(20:3(8Z,11Z,14Z)/14:1(9Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/14:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/14:0)

278

PC(14:0/20:2(11Z,14Z)), PC(14:1(9Z)/20:1(11Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(16:1(9Z)/18:1(9Z)), PC(18:1(11Z)/16:1(9Z)), PC(18:1(9Z)/16:1(9Z)), PC(18:2(9Z,12Z)/16:0), C42H80NO8P M+H PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PE(15:0/22:2(13Z,16Z)), PE(22:2(13Z,16Z)/15:0), Pe- NMe(18:1(9Z)/18:1(9Z)), Pe-NMe(16:1(9Z)/20:1(11Z)), Pe- NMe(18:1(9Z)/18:1(11Z)), Pe-NMe(18:1(11Z)/18:1(9Z)), Pe- NMe(18:1(11Z)/18:1(11Z)), Pe-NMe(14:0/22:2(13Z,16Z)), Pe- NMe(14:1(9Z)/22:1(13Z)), Pe-NMe(16:0/20:2(11Z,14Z)), Pe- NMe(18:0/18:2(9Z,12Z)), Pe-NMe(18:2(9Z,12Z)/18:0), Pe- NMe(20:1(11Z)/16:1(9Z)), Pe-NMe(20:2(11Z,14Z)/16:0), Pe- NMe(22:1(13Z)/14:1(9Z)), Pe-NMe(22:2(13Z,16Z)/14:0), Pe- NMe2(15:0/20:2(11Z,14Z)), Pe-NMe2(20:2(11Z,14Z)/15:0) PC(16:0/16:0), PC(14:0/18:0), PC(18:0/14:0), PE(15:0/20:0), C40H80NO8P M+Na PE(20:0/15:0), Pe-NMe(16:0/18:0), Pe-NMe(14:0/20:0), Pe- NMe(18:0/16:0), Pe-NMe(20:0/14:0), Pe-NMe2(15:0/18:0), Pe- NMe2(18:0/15:0)

279

PC(14:0/20:4(5Z,8Z,11Z,14Z)), PC(14:0/20:4(8Z,11Z,14Z,17Z)), PC(14:1(9Z)/20:3(5Z,8Z,11Z)), PC(14:1(9Z)/20:3(8Z,11Z,14Z)), PC(16:0/18:4(6Z,9Z,12Z,15Z)), PC(16:1(9Z)/18:3(6Z,9Z,12Z)), PC(16:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:3(6Z,9Z,12Z)/16:1(9Z)), PC(18:3(9Z,12Z,15Z)/16:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/16:0), PC(20:3(5Z,8Z,11Z)/14:1(9Z)), PC(20:3(8Z,11Z,14Z)/14:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/14:0), PC(20:4(8Z,11Z,14Z,17Z)/14:0), PE(15:0/22:4(7Z,10Z,13Z,16Z)), PE(22:4(7Z,10Z,13Z,16Z)/15:0), Pe- NMe(14:0/22:4(7Z,10Z,13Z,16Z)), Pe- NMe(16:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe(16:1(9Z)/20:3(5Z,8Z,11Z)), Pe- NMe(16:1(9Z)/20:3(8Z,11Z,14Z)), Pe- NMe(18:0/18:4(6Z,9Z,12Z,15Z)), Pe- C42H76NO8P M+H NMe(18:1(11Z)/18:3(6Z,9Z,12Z)), Pe- NMe(18:1(11Z)/18:3(9Z,12Z,15Z)), Pe- NMe(18:1(9Z)/18:3(6Z,9Z,12Z)), Pe- NMe(18:1(9Z)/18:3(9Z,12Z,15Z)), Pe- NMe(18:2(9Z,12Z)/18:2(9Z,12Z)), Pe- NMe(18:3(6Z,9Z,12Z)/18:1(11Z)), Pe- NMe(18:3(6Z,9Z,12Z)/18:1(9Z)), Pe- NMe(18:3(9Z,12Z,15Z)/18:1(11Z)), Pe- NMe(18:3(9Z,12Z,15Z)/18:1(9Z)), Pe- NMe(18:4(6Z,9Z,12Z,15Z)/18:0), Pe- NMe(20:3(5Z,8Z,11Z)/16:1(9Z)), Pe- NMe(20:3(8Z,11Z,14Z)/16:1(9Z)), Pe- NMe(20:4(5Z,8Z,11Z,14Z)/16:0), Pe- NMe(20:4(8Z,11Z,14Z,17Z)/16:0), Pe- NMe(22:4(7Z,10Z,13Z,16Z)/14:0), Pe- NMe2(15:0/20:4(5Z,8Z,11Z,14Z)), Pe- NMe2(15:0/20:4(8Z,11Z,14Z,17Z)), Pe- NMe2(20:4(5Z,8Z,11Z,14Z)/15:0), Pe- NMe2(20:4(8Z,11Z,14Z,17Z)/15:0), Pe- NMe(16:0/20:4(8Z,11Z,14Z,17Z))

280

PC(14:0/18:1(11Z)), PC(14:0/18:1(9Z)), PC(14:1(9Z)/18:0), PC(16:0/16:1(9Z)), PC(16:1(9Z)/16:0), PC(18:0/14:1(9Z)), PC(18:1(11Z)/14:0), PC(18:1(9Z)/14:0), PE(15:0/20:1(11Z)), PE(20:1(11Z)/15:0), Pe-NMe(16:0/18:1(9Z)), Pe- C40H78NO8P M+Na NMe(14:0/20:1(11Z)), Pe-NMe(14:1(9Z)/20:0), Pe- NMe(16:0/18:1(11Z)), Pe-NMe(16:1(9Z)/18:0), Pe- NMe(18:0/16:1(9Z)), Pe-NMe(18:1(11Z)/16:0), Pe- NMe(18:1(9Z)/16:0), Pe-NMe(20:0/14:1(9Z)), Pe- NMe(20:1(11Z)/14:0), Pe-NMe2(15:0/18:1(9Z)), Pe- NMe2(15:0/18:1(11Z)), Pe-NMe2(18:1(11Z)/15:0), Pe- NMe2(18:1(9Z)/15:0)

PC(14:0/18:2(9Z,12Z)), PC(14:1(9Z)/18:1(11Z)), PC(14:1(9Z)/18:1(9Z)), PC(16:1(9Z)/16:1(9Z)), PC(18:1(11Z)/14:1(9Z)), PC(18:1(9Z)/14:1(9Z)), PC(18:2(9Z,12Z)/14:0), PE(15:0/20:2(11Z,14Z)), C40H76NO8P M+Na PE(20:2(11Z,14Z)/15:0), Pe-NMe(14:1(9Z)/20:1(11Z)), Pe- NMe(16:1(9Z)/18:1(9Z)), Pe-NMe(16:1(9Z)/18:1(11Z)), Pe- NMe(14:0/20:2(11Z,14Z)), Pe-NMe(16:0/18:2(9Z,12Z)), Pe- NMe(18:1(9Z)/16:1(9Z)), Pe-NMe(18:1(11Z)/16:1(9Z)), Pe- NMe(18:2(9Z,12Z)/16:0), Pe-NMe(20:1(11Z)/14:1(9Z)), Pe- NMe(20:2(11Z,14Z)/14:0), Pe-NMe2(15:0/18:2(9Z,12Z)), Pe- NMe2(18:2(9Z,12Z)/15:0)

PE(14:0/20:2(11Z,14Z)), PE(14:1(9Z)/20:1(11Z)), PE(16:0/18:2(9Z,12Z)), PE(16:1(9Z)/18:1(11Z)), PE(16:1(9Z)/18:1(9Z)), PE(18:1(11Z)/16:1(9Z)), C39H74NO8P M+Na PE(18:1(9Z)/16:1(9Z)), PE(18:2(9Z,12Z)/16:0), PE(20:1(11Z)/14:1(9Z)), PE(20:2(11Z,14Z)/14:0), Pe- NMe(15:0/18:2(9Z,12Z)), Pe-NMe(18:2(9Z,12Z)/15:0), Pe- NMe2(14:1(9Z)/18:1(9Z)), Pe-NMe2(14:1(9Z)/18:1(11Z)), Pe- NMe2(16:1(9Z)/16:1(9Z)), Pe-NMe2(14:0/18:2(9Z,12Z)), Pe- NMe2(18:1(11Z)/14:1(9Z)), Pe-NMe2(18:1(9Z)/14:1(9Z)), Pe- NMe2(18:2(9Z,12Z)/14:0) C39H79N2O6P M+Na SM(d18:0/16:1(9Z)), Palmitoyl sphingomyelin C28H50NO7P M+Na LysoPC(20:4(5Z,8Z,11Z,14Z)), LysoPC(20:4(8Z,11Z,14Z,17Z)) C28H52NO7P M+H LysoPC(20:3(5Z,8Z,11Z)), LysoPC(20:3(8Z,11Z,14Z)) C26H54NO7P M+Na LysoPC(18:0), LysoPC(0:0/18:0), 2-acetyl-1-alkyl-sn-glycero-3- phosphocholine

281

C26H52NO7P M+Na LysoPC(18:1(9Z)), LysoPC(18:1(11Z)), 2-oleoyl-sn-glycero-3- phosphocholine C26H54NO7P M+H LysoPC(18:0), LysoPC(0:0/18:0), 2-acetyl-1-alkyl-sn-glycero-3- phosphocholine C26H48NO7P M+H LysoPC(18:3(6Z,9Z,12Z)), LysoPC(18:3(9Z,12Z,15Z)) C24H50NO7P M+Na LysoPC(16:0) TryptophanoI(R)-Boschniakine, 1-(2,3-Dihydro-1H-pyrrolizin- C10H11NO M+Na 5-yl)-2-propen-1-one, 3-[(5-Methyl-2-furanyl)methyl]-1H- pyrrole, 3,4-Dihydro-4-[(5-methyl-2-furanyl)methylene]-2H- pyrrole TG(16:0/18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), TG(16:1(9Z)/18:0/20:4(5Z,8Z,11Z,14Z)), TG(18:1(9Z)/18:2(9Z,12Z)/18:2(9Z,12Z)), TG(18:1(11Z)/16:0/20:4(5Z,8Z,11Z,14Z)), TG(18:1(9Z)/18:1(9Z)/18:3(6Z,9Z,12Z)), TG(18:1(9Z)/18:1(9Z)/18:3(9Z,12Z,15Z)), TG(18:2(9Z,12Z)/18:0/18:3(9Z,12Z,15Z)), TG(18:2(9Z,12Z)/18:1(11Z)/18:2(9Z,12Z)), Glycerol 1,2-di- (9Z,12Z-octadecadienoate) 3-(9Z-octadecenoate), Glycerol 1,3- di-(9Z,12Z-octadecadienoate) 2-(9Z-octadecenoate), TG(14:0/18:0/22:5(4Z,7Z,10Z,13Z,16Z)), TG(14:0/18:0/22:5(7Z,10Z,13Z,16Z,19Z)), TG(14:0/20:0/20:5(5Z,8Z,11Z,14Z,17Z)), TG(14:0/18:1(11Z)/22:4(7Z,10Z,13Z,16Z)), TG(14:0/18:1(9Z)/22:4(7Z,10Z,13Z,16Z)), TG(14:0/20:1(11Z)/20:4(5Z,8Z,11Z,14Z)), TG(14:0/20:1(11Z)/20:4(8Z,11Z,14Z,17Z)), C57H100O6 M+H TG(14:0/20:3(5Z,8Z,11Z)/20:2n6), TG(14:0/22:1(13Z)/18:4(6Z,9Z,12Z,15Z)), TG(14:0/18:3(6Z,9Z,12Z)/22:2(13Z,16Z)), TG(14:0/20:2n6/20:3(5Z,8Z,11Z)), TG(14:0/20:2n6/20:3n6), TG(14:0/20:3n6/20:2n6), TG(14:0/20:4(5Z,8Z,11Z,14Z)/20:1(11Z)), TG(14:0/22:2(13Z,16Z)/18:3(6Z,9Z,12Z)), TG(14:0/22:2(13Z,16Z)/18:3(9Z,12Z,15Z)), TG(14:0/22:4(7Z,10Z,13Z,16Z)/18:1(11Z)), TG(14:0/22:4(7Z,10Z,13Z,16Z)/18:1(9Z)), TG(14:0/22:5(4Z,7Z,10Z,13Z,16Z)/18:0), TG(14:0/18:3(9Z,12Z,15Z)/22:2(13Z,16Z)), TG(14:0/18:4(6Z,9Z,12Z,15Z)/22:1(13Z)), TG(14:0/20:4(8Z,11Z,14Z,17Z)/20:1(11Z)), TG(14:0/20:5(5Z,8Z,11Z,14Z,17Z)/20:0), TG(14:0/22:5(7Z,10Z,13Z,16Z,19Z)/18:0), TG(16:0/16:0/22:5(4Z,7Z,10Z,13Z,16Z)), TG(16:0/16:0/22:5(7Z,10Z,13Z,16Z,19Z)),

282

TG(16:0/18:0/20:5(5Z,8Z,11Z,14Z,17Z)), TG(16:0/16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), TG(16:0/18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), TG(16:0/18:1(11Z)/20:4(8Z,11Z,14Z,17Z)), TG(16:0/18:1(9Z)/20:4(8Z,11Z,14Z,17Z)), TG(16:0/20:1(11Z)/18:4(6Z,9Z,12Z,15Z)), TG(16:0/20:3(5Z,8Z,11Z)/18:2(9Z,12Z)), TG(16:0/18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), TG(16:0/18:2(9Z,12Z)/20:3n6), TG(16:0/18:3(6Z,9Z,12Z)/20:2n6), TG(16:0/20:2n6/18:3(6Z,9Z,12Z)), TG(16:0/20:2n6/18:3(9Z,12Z,15Z)), TG(16:0/20:3n6/18:2(9Z,12Z)), TG(16:0/20:4(5Z,8Z,11Z,14Z)/18:1(11Z))

PC(20:2(11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:3(5Z,8Z,11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:3(5Z,8Z,11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:3(8Z,11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:3(8Z,11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:4(7Z,10Z,13Z,16Z)), C50H84NO8P M+H PC(20:4(8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), PC(22:4(7Z,10Z,13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:4(7Z,10Z,13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:3(5Z,8Z,11Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:3(8Z,11Z,14Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:3(5Z,8Z,11Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z))

283

PC(20:3(5Z,8Z,11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:4(5Z,8Z,11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(20:4(8Z,11Z,14Z,17Z)/22:5(7Z,10Z,13Z,16Z,19Z)), C50H82NO8P M+H PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), PC(22:4(7Z,10Z,13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(5Z,8Z,11Z,14Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(5Z,8Z,11Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z))

284

PG(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(18:1(11Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PG(18:1(11Z)/22:5(7Z,10Z,13Z,16Z,19Z)), C46H79O10P M+Na PG(18:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PG(18:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PG(18:2(9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PG(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:2(11Z,14Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0) C41H82O13P2 M+H PGP(a-13:0/i-22:0), PGP(i-13:0/i-22:0) C47H95N2O6P M+Na SM(d18:0/24:1(15Z)) C57H86O4 M+H 3-Decaprenyl-4,5-dihydroxybenzoate

285

PC(16:1(9Z)/22:2(13Z,16Z)), PC(18:0/20:3(5Z,8Z,11Z)), PC(18:0/20:3(8Z,11Z,14Z)), PC(18:1(11Z)/20:2(11Z,14Z)), C46H86NO8P M+Na PC(18:1(9Z)/20:2(11Z,14Z)), PC(18:2(9Z,12Z)/20:1(11Z)), PC(18:3(6Z,9Z,12Z)/20:0), PC(18:3(9Z,12Z,15Z)/20:0), PC(20:0/18:3(6Z,9Z,12Z)), PC(20:0/18:3(9Z,12Z,15Z)), PC(20:1(11Z)/18:2(9Z,12Z)), PC(20:2(11Z,14Z)/18:1(11Z)), PC(20:2(11Z,14Z)/18:1(9Z)), PC(20:3(5Z,8Z,11Z)/18:0), PC(20:3(8Z,11Z,14Z)/18:0), PC(22:2(13Z,16Z)/16:1(9Z))

286

287

PC(18:3(6Z,9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:3(9Z,12Z,15Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(18:4(6Z,9Z,12Z,15Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PC(18:4(6Z,9Z,12Z,15Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PC(20:4(5Z,8Z,11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), C48H78NO8P M+H PC(20:4(8Z,11Z,14Z,17Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(5Z,8Z,11Z,14Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(8Z,11Z,14Z,17Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:4(6Z,9Z,12Z,15Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:3(6Z,9Z,12Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:3(9Z,12Z,15Z))

288

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), C44H84NO8P M+K PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine

289

PG(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PG(18:2(9Z,12Z)/22:5(4Z,7Z,10Z,13Z,16Z)), C46H77O10P M+H PG(18:2(9Z,12Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PG(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PG(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), PG(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PG(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z))

290

PE(20:3(5Z,8Z,11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(20:4(5Z,8Z,11Z,14Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(20:4(5Z,8Z,11Z,14Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(20:4(8Z,11Z,14Z,17Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(20:4(8Z,11Z,14Z,17Z)/22:5(7Z,10Z,13Z,16Z,19Z)), C47H76NO8P M+H PE(20:5(5Z,8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), PE(22:4(7Z,10Z,13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(5Z,8Z,11Z,14Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(5Z,8Z,11Z,14Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(5Z,8Z,11Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z))

291

292

PC(14:0/22:1(13Z)), PC(14:1(9Z)/22:0), PC(16:0/20:1(11Z)), C44H86NO8P M+Na PC(16:1(9Z)/20:0), PC(18:0/18:1(11Z)), PC(18:0/18:1(9Z)), PC(18:1(11Z)/18:0), PC(18:1(9Z)/18:0), PC(20:0/16:1(9Z)), PC(20:1(11Z)/16:0), PC(22:0/14:1(9Z)), PC(22:1(13Z)/14:0), PE(15:0/24:1(15Z)), PE(24:1(15Z)/15:0)

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), C44H84NO8P M+Na PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine

293

294

295

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(16:0/18:1(11Z)), C42H82NO8P M+K PC(16:0/18:1(9Z)), PC(16:1(9Z)/18:0), PC(18:0/16:1(9Z)), PC(18:1(11Z)/16:0), PC(18:1(9Z)/16:0), PC(20:0/14:1(9Z)), PC(20:1(11Z)/14:0), PE(15:0/22:1(13Z)), PE(22:1(13Z)/15:0)

PG(16:0/22:5(4Z,7Z,10Z,13Z,16Z)), PG(16:0/22:5(7Z,10Z,13Z,16Z,19Z)), PG(16:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PG(18:1(11Z)/20:4(5Z,8Z,11Z,14Z)), C44H77O10P M+H PG(18:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PG(18:2(9Z,12Z)/20:3(5Z,8Z,11Z)), PG(18:2(9Z,12Z)/20:3(8Z,11Z,14Z)), PG(20:3(8Z,11Z,14Z)/18:2(9Z,12Z)), PG(20:4(5Z,8Z,11Z,14Z)/18:1(9Z)), PG(22:5(7Z,10Z,13Z,16Z,19Z)/16:0) PG(18:1(11Z)/18:1(9Z)), PG(18:0/18:2(9Z,12Z)), C42H79O10P M+Na PG(18:1(11Z)/18:1(11Z)), PG(18:1(9Z)/18:1(11Z)), PG(18:1(9Z)/18:1(9Z)), PG(18:2(9Z,12Z)/18:0)

296

297

PC(15:0/20:2(11Z,14Z)), PC(20:2(11Z,14Z)/15:0), PE(14:1(9Z)/24:1(15Z)), PE(16:0/22:2(13Z,16Z)), PE(16:1(9Z)/22:1(13Z)), PE(18:0/20:2(11Z,14Z)), C43H82NO8P M+Na PE(18:1(11Z)/20:1(11Z)), PE(18:1(9Z)/20:1(11Z)), PE(18:2(9Z,12Z)/20:0), PE(20:0/18:2(9Z,12Z)), PE(20:1(11Z)/18:1(11Z)), PE(20:1(11Z)/18:1(9Z)), PE(20:2(11Z,14Z)/18:0), PE(22:1(13Z)/16:1(9Z)), PE(22:2(13Z,16Z)/16:0), PE(24:1(15Z)/14:1(9Z)), PE- NMe2(18:1(9Z)/18:1(9Z))

298

299

PC(18:1(9Z)/18:1(9Z)), PC(14:0/22:2(13Z,16Z)), PC(14:1(9Z)/22:1(13Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(18:0/18:2(9Z,12Z)), C44H84NO8P M+H PC(18:1(11Z)/18:1(11Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PC(18:2(9Z,12Z)/18:0), PC(20:1(11Z)/16:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:1(13Z)/14:1(9Z)), PC(22:2(13Z,16Z)/14:0), 1,2-dioleoyl- sn-glycero-3-phosphocholine

300

PE(18:3(6Z,9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:3(9Z,12Z,15Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:5(4Z,7Z,10Z,13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/22:5(7Z,10Z,13Z,16Z,19Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), C45H72NO8P M+H PE(20:4(8Z,11Z,14Z,17Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(8Z,11Z,14Z,17Z)), PE(22:5(4Z,7Z,10Z,13Z,16Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:5(7Z,10Z,13Z,16Z,19Z)/18:4(6Z,9Z,12Z,15Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:3(6Z,9Z,12Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:3(9Z,12Z,15Z))

C42H84NO8P M+Na PC(14:0/20:0), PC(16:0/18:0), PC(18:0/16:0), PC(20:0/14:0), PE(15:0/22:0), PE(22:0/15:0), PE-NMe(18:0/18:0)

301

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(16:0/18:1(11Z)), C42H82NO8P M+Na PC(16:0/18:1(9Z)), PC(16:1(9Z)/18:0), PC(18:0/16:1(9Z)), PC(18:1(11Z)/16:0), PC(18:1(9Z)/16:0), PC(20:0/14:1(9Z)), PC(20:1(11Z)/14:0), PE(15:0/22:1(13Z)), PE(22:1(13Z)/15:0)

302

PC(14:0/22:5(4Z,7Z,10Z,13Z,16Z)), PC(14:0/22:5(7Z,10Z,13Z,16Z,19Z)), PC(14:1(9Z)/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:5(5Z,8Z,11Z,14Z,17Z)), PC(16:1(9Z)/20:4(5Z,8Z,11Z,14Z)), PC(16:1(9Z)/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/18:4(6Z,9Z,12Z,15Z)), PC(18:1(9Z)/18:4(6Z,9Z,12Z,15Z)), C44H78NO8P M+H PC(18:2(9Z,12Z)/18:3(6Z,9Z,12Z)), PC(18:2(9Z,12Z)/18:3(9Z,12Z,15Z)), PC(18:3(6Z,9Z,12Z)/18:2(9Z,12Z)), PC(18:3(9Z,12Z,15Z)/18:2(9Z,12Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:1(9Z)), PC(20:4(8Z,11Z,14Z,17Z)/16:1(9Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:1(9Z)), PC(22:5(4Z,7Z,10Z,13Z,16Z)/14:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/14:0)

303

PC(15:0/18:1(11Z)), PC(15:0/18:1(9Z)), PC(18:1(11Z)/15:0), PC(18:1(9Z)/15:0), PE(14:0/22:1(13Z)), PE(14:1(9Z)/22:0), C41H80NO8P M+Na PE(16:0/20:1(11Z)), PE(16:1(9Z)/20:0), PE(18:0/18:1(11Z)), PE(18:0/18:1(9Z)), PE(18:1(11Z)/18:0), PE(18:1(9Z)/18:0), PE(20:0/16:1(9Z)), PE(20:1(11Z)/16:0), PE(22:0/14:1(9Z)), PE(22:1(13Z)/14:0), PE-NMe2(16:0/18:1(9Z))

304

PC(15:0/18:2(9Z,12Z)), PC(18:2(9Z,12Z)/15:0), PE(14:0/22:2(13Z,16Z)), PE(14:1(9Z)/22:1(13Z)), PE(16:0/20:2(11Z,14Z)), PE(16:1(9Z)/20:1(11Z)), C41H78NO8P M+Na PE(18:0/18:2(9Z,12Z)), PE(18:1(11Z)/18:1(11Z)), PE(18:1(11Z)/18:1(9Z)), PE(18:1(9Z)/18:1(11Z)), PE(18:1(9Z)/18:1(9Z)), PE(18:2(9Z,12Z)/18:0), PE(20:1(11Z)/16:1(9Z)), PE(20:2(11Z,14Z)/16:0), PE(22:1(13Z)/14:1(9Z)), PE(22:2(13Z,16Z)/14:0)

305

PE(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(18:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:3(6Z,9Z,12Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:4(8Z,11Z,14Z,17Z)), PE(18:3(9Z,12Z,15Z)/20:4(5Z,8Z,11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:4(8Z,11Z,14Z,17Z)), C43H72NO8P M+H PE(18:4(6Z,9Z,12Z,15Z)/20:3(5Z,8Z,11Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:3(8Z,11Z,14Z)), PE(20:3(5Z,8Z,11Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:3(8Z,11Z,14Z)/18:4(6Z,9Z,12Z,15Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:3(6Z,9Z,12Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:3(9Z,12Z,15Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:3(6Z,9Z,12Z)), PE(20:4(8Z,11Z,14Z,17Z)/18:3(9Z,12Z,15Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/18:2(9Z,12Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/16:1(9Z))

306

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(16:0/18:1(11Z)), C42H82NO8P M+H PC(16:0/18:1(9Z)), PC(16:1(9Z)/18:0), PC(18:0/16:1(9Z)), PC(18:1(11Z)/16:0), PC(18:1(9Z)/16:0), PC(20:0/14:1(9Z)), PC(20:1(11Z)/14:0), PE(15:0/22:1(13Z)), PE(22:1(13Z)/15:0)

PC(14:0/18:1(11Z)), PC(14:0/18:1(9Z)), PC(14:1(9Z)/18:0), C40H78NO8P M+Na PC(16:0/16:1(9Z)), PC(16:1(9Z)/16:0), PC(18:0/14:1(9Z)), PC(18:1(11Z)/14:0), PC(18:1(9Z)/14:0), PE(15:0/20:1(11Z)), PE(20:1(11Z)/15:0), PE-NMe(16:0/18:1(9Z)) C39H79N2O6P M+Na SM(d18:0/16:1(9Z)), Palmitoyl sphingomyelin C28H52NO7P M+H LysoPC(20:3(5Z,8Z,11Z)), LysoPC(20:3(8Z,11Z,14Z)) C26H54NO7P M+Na LysoPC(18:0), LysoPC(0:0/18:0)

307

C26H52NO7P M+Na LysoPC(18:1(9Z)), LysoPC(18:1(11Z)), 2-oleoyl-sn-glycero-3- phosphocholine C26H54NO7P M+H LysoPC(18:0), LysoPC(0:0/18:0) C26H48NO7P M+H LysoPC(18:3(6Z,9Z,12Z)), LysoPC(18:3(9Z,12Z,15Z)) C24H50NO7P M+Na LysoPC(16:0) PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:3(8Z,11Z,14Z)), C50H82NO8P M+H PC(20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:5(5Z,8Z,11Z,14Z,17Z)/22:4(7Z,10Z,13Z,16Z)), PC(22:4(7Z,10Z,13Z,16Z)/20:5(5Z,8Z,11Z,14Z,17Z)) C44H83NO13 M+Na LacCer(d18:1/14:0) C42H84NO12P M+Na PI-Cer(t18:0/18:0), PI-Cer(t20:0/16:0), PI- Cer(d18:0/18:0(2OH)), PI-Cer(d20:0/16:0(2OH))

PC(16:0/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:4(8Z,10Z,12Z,14Z)), PC(18:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PC(20:1(11Z)/18:3(9Z,12Z,15Z)), C46H84NO8P M+Na PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(22:4(7Z,10Z,13Z,16Z)/16:0), PC(18:1(11Z)/20:3(5Z,8Z,11Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/18:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/21:0), PE(22:4(7Z,10Z,13Z,16Z)/19:0), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:1(11Z)), PC(20:1(11Z)/18:3(6Z,9Z,12Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PC(20:3(5Z,8Z,11Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PE(19:0/22:4(7Z,10Z,13Z,16Z)), PE(21:0/20:4(5Z,8Z,11Z,14Z))

308

PC(14:0/22:2(13Z,16Z)), PC(19:0/17:2(9Z,12Z)), PE(19:1(9Z)/20:1(11Z)), PC(18:1(17Z)/18:1(17Z)), PC(18:0/18:2(6Z,9Z)), PC(18:1(11E)/18:1(11E)), PC(18:1(13Z)/18:1(13Z)), PC(18:1(14Z)/18:1(14Z)), PC(18:1(15Z)/18:1(15Z)), PC(18:1(16Z)/18:1(16Z)), PC(18:1(2Z)/18:1(2Z)), PC(18:1(5Z)/18:1(5Z)), PC(18:1(9Z)/18:1(9Z)), PC(18:1(4Z)/18:1(4Z)), PC(14:1(9Z)/22:1(11Z)), PC(16:0/20:2(11Z,14Z)), PC(16:1(9Z)/20:1(11Z)), PC(17:2(9Z,12Z)/19:0), PC(19:1(9Z)/17:1(9Z)), PC(20:1(11Z)/16:1(9Z)), C44H84NO8P M+K PC(22:1(11Z)/14:1(9Z)), PC(22:1(13Z)/14:1(9Z)), PE(20:1(11Z)/19:1(9Z)), PE(21:0/18:2(9Z,12Z)), PE(22:0/17:2(9Z,12Z)), PC(18:0/18:2(10Z,12Z)), PC(18:0/18:2(2E,4E)), PC(18:0/18:2(9Z,12Z)), PC(18:1(10Z)/18:1(10Z)), PC(18:1(11Z)/18:1(11Z)), PC(18:1(12Z)/18:1(12Z)), PC(18:1(3Z)/18:1(3Z)), PC(18:1(6E)/18:1(6E)), PC(18:1(6Z)/18:1(6Z)), PC(18:1(7Z)/18:1(7Z)), PC(18:1(8Z)/18:1(8Z)), PC(18:1(9E)/18:1(9E)), PC(18:2(9Z,12Z)/18:0), PC(17:1(9Z)/19:1(9Z)), PC(20:2(11Z,14Z)/16:0), PC(22:2(13Z,16Z)/14:0), PC(14:1(9Z)/22:1(13Z)), PC(18:1(11Z)/18:1(9Z)), PC(18:1(9Z)/18:1(11Z)), PE(17:0/22:2(13Z,16Z)), PE(17:1(9Z)/22:1(11Z)), PE(17:2(9Z,12Z)/22:0), PE(18:2(9Z,12Z)/21:0), PE(19:0/20:2(11Z,14Z)), PE(20:2(11Z,14Z)/19:0)

309

PC(16:0/22:4(7Z,10Z,13Z,16Z)), PC(18:0/20:4(8Z,10Z,12Z,14Z)), PC(18:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:2(9Z,12Z)/20:2(11Z,14Z)), PC(18:3(6Z,9Z,12Z)/20:1(11Z)), PC(18:4(6Z,9Z,12Z,15Z)/20:0), PC(20:0/18:4(6Z,9Z,12Z,15Z)), PC(20:1(11Z)/18:3(9Z,12Z,15Z)), C46H84NO8P M+H PC(20:3(8Z,11Z,14Z)/18:1(9Z)), PC(22:4(7Z,10Z,13Z,16Z)/16:0), PC(18:1(11Z)/20:3(5Z,8Z,11Z)), PC(18:1(11Z)/20:3(8Z,11Z,14Z)), PC(20:3(5Z,8Z,11Z)/18:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/21:0), PE(22:4(7Z,10Z,13Z,16Z)/19:0), PC(18:0/20:4(5Z,8Z,11Z,14Z)), PC(18:3(9Z,12Z,15Z)/20:1(11Z)), PC(20:1(11Z)/18:3(6Z,9Z,12Z)), PC(20:2(11Z,14Z)/18:2(9Z,12Z)), PC(20:4(5Z,8Z,11Z,14Z)/18:0), PC(18:0/20:4(8Z,11Z,14Z,17Z)), PC(20:3(5Z,8Z,11Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/18:1(11Z)), PE(19:0/22:4(7Z,10Z,13Z,16Z)), PE(21:0/20:4(5Z,8Z,11Z,14Z))

PC(14:0/22:1(11Z)), PC(18:0/18:1(13Z)), PC(18:0/18:1(7Z)), PC(18:1(9Z)/18:0), PC(14:1(9Z)/22:0), PC(15:1(9Z)/21:0), PC(16:0/20:1(11Z)), PC(17:0/19:1(9Z)), PC(19:0/17:1(9Z)), PC(19:1(9Z)/17:0), PC(20:1(11Z)/16:0), PC(22:1(11Z)/14:0), C44H86NO8P M+Na PC(14:0/22:1(13Z)), PE(17:0/22:1(11Z)), PE(17:1(9Z)/22:0), PE(19:1(9Z)/20:0), PE(20:1(11Z)/19:0), PE(22:0/17:1(9Z)), PE(22:1(11Z)/17:0), PC(18:0/18:1(16Z)), PC(18:0/18:1(6Z)), PC(18:0/18:1(11Z)), PC(18:0/18:1(12Z)), PC(18:0/18:1(9E)), PC(18:0/18:1(9Z)), PC(18:1(11Z)/18:0), PC(16:1(9Z)/20:0), PC(17:1(9Z)/19:0), PC(20:0/16:1(9Z)), PC(21:0/15:1(9Z)), PC(22:0/14:1(9Z)), PC(22:1(13Z)/14:0), PE(18:1(9Z)/21:0), PE(19:0/20:1(11Z)), PE(20:0/19:1(9Z)), PE(21:0/18:1(9Z))

310

PC(14:0/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:4(5E,8E,11E,14E)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PC(18:0/18:4(9E,11E,13E,15E)), PC(18:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(6Z,9Z)/18:2(6Z,9Z)), PC(18:2(9Z,11Z)/18:2(9Z,11Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(16:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/16:1(9Z)), PE(17:0/22:4(7Z,10Z,13Z,16Z)), PE(19:1(9Z)/20:3(8Z,11Z,14Z)), PE(22:4(7Z,10Z,13Z,16Z)/17:0), C44H80NO8P M+Na PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(18:2(2E,4E)/18:2(2E,4E)), PC(18:2(2Z,4Z)/18:2(2Z,4Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PC(20:4(8E,11E,14E,17E)/16:0), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(16:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/18:3(6Z,9Z,12Z)), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PE(17:2(9Z,12Z)/22:2(13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/21:0), PE(19:0/20:4(5Z,8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/19:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/19:0), PE(21:0/18:4(6Z,9Z,12Z,15Z)), PE(22:2(13Z,16Z)/17:2(9Z,12Z))

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(19:0/15:1(9Z)), PC(19:1(9Z)/15:0), PC(16:0/18:1(6Z)), PC(16:0/18:1(9E)), PC(16:0/18:1(6E)), PC(16:0/18:1(11Z)), PC(12:0/22:1(11Z)), PC(15:0/19:1(9Z)), PC(20:0/14:1(9Z)), PC(22:1(11Z)/12:0), C42H82NO8P M+K PE(17:0/20:1(11Z)), PE(18:1(9Z)/19:0), PE(19:0/18:1(9Z)), PE(19:1(9Z)/18:0), PE(20:0/17:1(9Z)), PE(20:1(11Z)/17:0), PE(21:0/16:1(9Z)), PE(22:0/15:1(9Z)), PC(16:0/18:1(11E)), PC(16:0/18:1(9Z)), PC(18:0/16:1(9Z)), PC(18:1(9Z)/16:0), PC(15:1(9Z)/19:0), PC(16:1(9Z)/18:0), PC(17:0/17:1(9Z)), PC(17:1(9Z)/17:0), PC(20:1(11Z)/14:0), PC(18:1(11Z)/16:0), PE(15:0/22:1(11Z)), PE(15:1(9Z)/22:0), PE(16:1(9Z)/21:0), PE(17:1(9Z)/20:0), PE(18:0/19:1(9Z)), PE(22:1(11Z)/15:0) C45H78NO7P M+Na PE(P-18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))

311

PG(17:1(9Z)/19:1(9Z)), PG(17:2(9Z,12Z)/19:0), PG(19:1(9Z)/17:1(9Z)), PG(20:1(11Z)/16:1(9Z)), PG(20:2(11Z,14Z)/16:0), PG(22:2(13Z,16Z)/14:0), C42H79O10P M+Na PG(18:2(9Z,12Z)/18:0), PG(18:0/18:2(9Z,12Z)), PG(18:1(9E)/18:1(9E)), PG(14:0/22:2(13Z,16Z)), PG(14:1(9Z)/22:1(11Z)), PG(16:0/20:2(11Z,14Z)), PG(16:1(9Z)/20:1(11Z)), PG(19:0/17:2(9Z,12Z)), PG(22:1(11Z)/14:1(9Z)), PG(18:1(9Z)/18:1(9Z)), LBPA(18:1(9Z)/18:1(9Z))

PC(15:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(17:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(18:3(6Z,9Z,12Z)/22:4(7Z,10Z,13Z,16Z)), PE(18:3(9Z,12Z,15Z)/22:4(7Z,10Z,13Z,16Z)), PE(20:4(5Z,8Z,11Z,14Z)/20:3(8Z,11Z,14Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:3(9Z,12Z,15Z)), C45H76NO8P M+H PC(20:5(5Z,8Z,11Z,14Z,17Z)/17:2(9Z,12Z)), PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/15:1(9Z)), PE(20:2(11Z,14Z)/20:5(5Z,8Z,11Z,14Z,17Z)), PE(20:3(8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)), PE(20:5(5Z,8Z,11Z,14Z,17Z)/20:2(11Z,14Z)), PE(22:4(7Z,10Z,13Z,16Z)/18:3(6Z,9Z,12Z)), PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:1(9Z)), PE(18:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z))

312

PC(15:0/20:4(5Z,8Z,11Z,14Z)), PC(18:2(9Z,12E)/17:2(9Z,11E)), PC(13:0/22:4(7Z,10Z,13Z,16Z)), PC(15:1(9Z)/20:3(8Z,11Z,14Z)), PC(17:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(9Z,12Z)/17:2(9Z,12Z)), PC(20:3(8Z,11Z,14Z)/15:1(9Z)), PC(20:4(5Z,8Z,11Z,14Z)/15:0), PC(22:4(7Z,10Z,13Z,16Z)/13:0), PE(18:1(9Z)/20:3(8Z,11Z,14Z)), PE(18:3(6Z,9Z,12Z)/20:1(11Z)), PE(20:3(8Z,11Z,14Z)/18:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/18:0), PE(P- 18:0/20:4(5Z,8Z,10E,14Z)(12OH[S])), PC(17:0/18:4(6Z,9Z,12Z,15Z)), PC(17:1(9Z)/18:3(6Z,9Z,12Z)), C43H78NO8P M+Na PC(17:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(6Z,9Z,12Z)/17:1(9Z)), PC(18:3(9Z,12Z,15Z)/17:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/17:0), PE(20:0/18:4(6Z,9Z,12Z,15Z)), PE(18:1(9Z)/20:3(5Z,8Z,11Z)), PE(16:0/22:4(7Z,10Z,13Z,16Z)), PE(18:0/20:4(5Z,8Z,11Z,14Z)), PE(18:0/20:4(5E,8E,11E,14E)), PE(18:2(9Z,12Z)/20:2(11Z,14Z)), PE(18:3(9Z,12Z,15Z)/20:1(11Z)), PE(18:4(6Z,9Z,12Z,15Z)/20:0), PE(20:1(11Z)/18:3(6Z,9Z,12Z)), PE(20:1(11Z)/18:3(9Z,12Z,15Z)), PE(20:2(11Z,14Z)/18:2(9Z,12Z)), PE(22:4(7Z,10Z,13Z,16Z)/16:0), PE(P- 18:0/20:4(5Z,8Z,11Z,13E)(15OH[S])), PE(P- 18:0/20:4(6E,8Z,11Z,14Z)(5OH[S]))

313

PC(14:0/22:4(7Z,10Z,13Z,16Z)), PC(16:0/20:4(5E,8E,11E,14E)), PC(18:0/18:4(6Z,9Z,12Z,15Z)), PC(18:0/18:4(9E,11E,13E,15E)), PC(18:1(9Z)/18:3(9Z,12Z,15Z)), PC(18:2(6Z,9Z)/18:2(6Z,9Z)), PC(18:2(9Z,11Z)/18:2(9Z,11Z)), PC(20:4(5Z,8Z,11Z,14Z)/16:0), PC(16:1(9Z)/20:3(8Z,11Z,14Z)), PC(18:1(9Z)/18:3(6Z,9Z,12Z)), PC(20:3(8Z,11Z,14Z)/16:1(9Z)), PC(16:1(9Z)/20:3(5Z,8Z,11Z)), PC(18:3(6Z,9Z,12Z)/18:1(11Z)), PC(20:3(5Z,8Z,11Z)/16:1(9Z)), PE(17:0/22:4(7Z,10Z,13Z,16Z)), PE(19:1(9Z)/20:3(8Z,11Z,14Z)), PE(22:4(7Z,10Z,13Z,16Z)/17:0), C44H80NO8P M+H PC(16:0/20:4(5Z,8Z,11Z,14Z)), PC(18:2(2E,4E)/18:2(2E,4E)), PC(18:2(2Z,4Z)/18:2(2Z,4Z)), PC(18:2(9Z,12Z)/18:2(9Z,12Z)), PC(18:3(9Z,12Z,15Z)/18:1(9Z)), PC(20:4(8E,11E,14E,17E)/16:0), PC(18:3(6Z,9Z,12Z)/18:1(9Z)), PC(18:4(6Z,9Z,12Z,15Z)/18:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(16:0/20:4(8Z,11Z,14Z,17Z)), PC(18:1(11Z)/18:3(6Z,9Z,12Z)), PC(18:1(11Z)/18:3(9Z,12Z,15Z)), PC(18:3(9Z,12Z,15Z)/18:1(11Z)), PE(17:2(9Z,12Z)/22:2(13Z,16Z)), PE(18:4(6Z,9Z,12Z,15Z)/21:0), PE(19:0/20:4(5Z,8Z,11Z,14Z)), PE(20:3(8Z,11Z,14Z)/19:1(9Z)), PE(20:4(5Z,8Z,11Z,14Z)/19:0), PE(21:0/18:4(6Z,9Z,12Z,15Z)), PE(22:2(13Z,16Z)/17:2(9Z,12Z))

PC(14:0/20:1(11Z)), PC(14:1(9Z)/20:0), PC(19:0/15:1(9Z)), PC(19:1(9Z)/15:0), PC(16:0/18:1(6Z)), PC(16:0/18:1(9E)), PC(16:0/18:1(6E)), PC(16:0/18:1(11Z)), PC(12:0/22:1(11Z)), PC(15:0/19:1(9Z)), PC(20:0/14:1(9Z)), PC(22:1(11Z)/12:0), C42H82NO8P M+Na PE(17:0/20:1(11Z)), PE(18:1(9Z)/19:0), PE(19:0/18:1(9Z)), PE(19:1(9Z)/18:0), PE(20:0/17:1(9Z)), PE(20:1(11Z)/17:0), PE(21:0/16:1(9Z)), PE(22:0/15:1(9Z)), PC(16:0/18:1(11E)), PC(16:0/18:1(9Z)), PC(18:0/16:1(9Z)), PC(18:1(9Z)/16:0), PC(15:1(9Z)/19:0), PC(16:1(9Z)/18:0), PC(17:0/17:1(9Z)), PC(17:1(9Z)/17:0), PC(20:1(11Z)/14:0), PC(18:1(11Z)/16:0), PE(15:0/22:1(11Z)), PE(15:1(9Z)/22:0), PE(16:1(9Z)/21:0), PE(17:1(9Z)/20:0), PE(18:0/19:1(9Z)), PE(22:1(11Z)/15:0)

314

PC(14:0/20:2(11Z,14Z)), PE-NMe(18:1(9E)/18:1(9E)), PE- NMe(18:1(9Z)/18:1(9Z)), PE(20:0/17:2(9Z,12Z)), PC(16:0/18:2(11Z,13Z)), PC(16:0/18:2(9E,11E)), PC(16:0/18:2(9E,11Z)), PC(16:0/18:2(9E,12E)), PC(16:1(2Z)/18:1(9Z)), PC(16:1(9Z)/18:1(9Z)), PC(17:1(9Z)/17:1(9Z)), PC(18:0/16:2(2E,4E)), PC(18:1(9Z)/16:1(9Z)), PC(18:2(2E,4E)/16:0), PC(18:2(6Z,9Z)/16:0), PC(12:0/22:2(13Z,16Z)), PC(14:1(9Z)/20:1(11Z)), PC(17:0/17:2(9Z,12Z)), C42H80NO8P M+Na PC(17:2(9Z,12Z)/17:0), PC(20:1(11Z)/14:1(9Z)), PC(20:2(11Z,14Z)/14:0), PC(18:1(11Z)/16:1(9Z)), PE(15:0/22:2(13Z,16Z)), PE(17:0/20:2(11Z,14Z)), PE(19:1(9Z)/18:1(9Z)), PE(20:1(11Z)/17:1(9Z)), PE(20:2(11Z,14Z)/17:0), PE(22:1(11Z)/15:1(9Z)), PE(22:2(13Z,16Z)/15:0), PC(16:0/18:2(10E,12Z)), PC(16:0/18:2(2E,4E)), PC(16:0/18:2(2Z,4Z)), PC(16:0/18:2(6Z,9Z)), PC(16:0/18:2(9Z,12Z)), PC(16:1(9Z)/18:1(11Z)), PC(17:1(10Z)/17:1(10Z)), PC(18:2(9Z,12Z)/16:0), PC(15:1(9Z)/19:1(9Z)), PC(19:1(9Z)/15:1(9Z)), PC(22:2(13Z,16Z)/12:0), PE(15:1(9Z)/22:1(11Z)), PE(17:1(9Z)/20:1(11Z)), PE(17:2(9Z,12Z)/20:0), PE(18:1(9Z)/19:1(9Z)), PE(18:2(9Z,12Z)/19:0), PE(19:0/18:2(9Z,12Z))

PC(15:0/17:1(9Z)), PE-NMe(16:0/18:1(9Z)), PE- NMe(18:1(9Z)/16:0), PE(19:1(9Z)/16:0), PE(20:0/15:1(9Z)), PC(12:0/20:1(11Z)), PC(17:1(9Z)/15:0), PC(14:0/18:1(9Z)), PC(16:0/16:1(9Z)), PC(13:0/19:1(9Z)), PC(14:1(9Z)/18:0), C40H78NO8P M+Na PC(15:1(9Z)/17:0), PC(18:0/14:1(9Z)), PC(18:1(11Z)/14:0), PC(19:1(9Z)/13:0), PC(20:1(11Z)/12:0), PE(13:0/22:1(11Z)), PE(16:0/19:1(9Z)), PE(17:1(9Z)/18:0), PE(18:0/17:1(9Z)), PE(20:1(11Z)/15:0), PE(17:0/18:1(9Z)), PC(14:0/18:1(11Z)), PC(18:1(9Z)/14:0), PC(16:1(9Z)/16:0), PC(17:0/15:1(9Z)), PE(14:1(9Z)/21:0), PE(15:0/20:1(11Z)), PE(15:1(9Z)/20:0), PE(16:1(9Z)/19:0), PE(18:1(9Z)/17:0), PE(19:0/16:1(9Z)), PE(21:0/14:1(9Z)), PE(22:1(11Z)/13:0), PE(16:0/18:0(11Cp))

C39H79N2O6P M+Na SM(d16:1/18:0), SM(d17:1/17:0), PE-Cer(d14:1(4E)/23:0), PE- Cer(d16:1(4E)/21:0), SM(d18:1/16:0), PE-Cer(d15:1(4E)/22:0)

315

Appendix C

Supplemental Information for Chapter 4 Table C.1. One hundred most abundant background ions detected from ESI solvent (0.2% formic acid for +ve and 1 mM acetic acid for -ve mode).

background ions (100 most abundant) 150-600, +ve ESI 150-600, -ve ESI 500-2000, +ve ESI 500-2000, -ve ESI 217.1069 255.2330 637.3068 695.3134 217.1072 255.2325 637.3087 695.3112 371.1014 255.2335 637.3049 681.2977 371.1006 283.2645 503.1095 695.3156 217.1065 283.2639 637.3106 681.2956 223.0636 255.2320 519.1412 695.3090 223.0632 283.2650 536.1664 681.2999 173.0809 255.2339 536.1679 681.2935 217.1076 283.2633 503.1081 695.3177 371.1023 283.2656 519.1398 696.3165 223.0640 157.1223 503.1108 696.3144 371.0997 157.1226 610.1858 681.3020 173.0811 157.1221 637.3030 696.3187 173.0806 255.2315 519.1426 682.3003 355.0701 256.2361 536.1650 695.3069 355.0693 171.1381 610.1876 682.3024 161.0960 255.2344 536.1694 682.2982 161.0963 256.2366 638.3114 696.3122 223.0628 283.2628 550.2191 681.2914 217.1061 157.1228 638.3095 696.3209 183.0803 171.1379 610.1841 682.3045 183.0806 171.1384 519.1384 695.3199 391.2844 284.2676 503.1068 682.2961 391.2835 256.2356 550.2176 681.3041 355.0709 199.1698 550.2206 561.4866 161.0958 199.1695 638.3133 561.4881 371.1031 157.1219 654.3338 696.3100 342.1853 227.2012 503.1121 695.3047 355.0685 284.2681 637.3125 561.4850 173.0814 256.2371 519.1440 696.3231 163.0754 227.2016 624.2384 653.3032 223.0644 283.2662 610.1894 533.4555

316

173.0803 284.2670 638.3076 656.4324 342.1845 227.2008 520.1398 682.3066 217.1080 199.1702 624.2365 653.3012 163.0751 171.1376 654.3357 656.4344 161.0965 199.1692 611.1861 533.4569 266.8881 171.1387 520.1412 561.4897 266.8886 284.2687 537.1673 681.2893 280.9040 220.1465 654.3318 533.4540 280.8677 256.2352 504.1091 697.3197 183.0800 157.1230 610.1823 656.4304 371.0989 171.1018 537.1658 653.3052 370.2164 220.1461 504.1078 891.5328 235.1693 241.2173 536.1635 682.2940 235.1689 227.2020 624.2402 653.2993 177.1637 241.2168 536.1708 891.5297 342.1860 284.2664 520.1384 697.3175 171.1378 171.1015 550.2222 697.3219 183.0809 220.1469 611.1879 656.4364 177.1639 255.2310 550.2161 561.4834 391.2853 227.2004 684.2060 533.4584 171.1381 311.1689 519.1370 891.5360 163.0756 255.2349 684.2039 628.4014 280.8671 157.1216 611.1843 695.3221 391.2825 171.1020 577.1282 628.4033 249.1846 199.1705 537.1688 698.2878 280.9034 297.2435 504.1104 533.4526 163.0605 241.2177 637.3011 628.3995 370.2155 283.2622 520.1426 891.5265 370.2172 256.2376 593.1602 698.2856 194.9407 311.1682 521.1370 641.4059 155.0703 325.1844 638.3152 697.3153 280.9045 311.1695 626.4578 683.3029 236.8778 220.1458 593.1585 656.4284 316.2114 241.2164 537.1644 641.4078 249.1850 199.1688 505.1063 561.4913 371.3151 297.2441 577.1266 683.3051 280.8683 233.1546 626.4596 653.3072 316.2121 213.1857 504.1064 698.2900 163.0391 185.1539 654.3377 743.1669

317

223.0625 213.1853 538.1636 743.1645 163.0603 325.1850 624.2347 697.3240 342.1838 233.1542 503.1055 743.1693 192.1382 299.2592 505.1561 681.3062 445.1193 171.1373 577.1299 743.1621 445.1205 297.2429 551.2197 919.5645 163.0388 171.1013 551.2182 683.3008 155.0701 157.0859 612.1834 641.4040 387.1799 284.2693 521.1384 743.1861 372.1002 157.0861 654.3298 628.4051 371.3160 325.1837 684.2081 653.2973 266.8875 220.1473 625.2381 919.5612 194.9404 185.1536 612.1816 891.5391 177.1634 283.2667 521.1356 743.1885 235.1697 185.1542 505.1050 743.1837 161.0955 227.2024 538.1621 696.3078 271.2629 171.1023 638.3056 641.4098 266.8891 158.1258 522.2291 682.3087 299.0613 269.2122 684.2017 743.1597 355.0717 299.2598 611.1896 743.1717 257.2472 171.1389 505.1575 743.1909 171.1376 253.2175 505.1077 743.1933 163.0749 299.2586 538.1651 919.5678 235.1684 269.2127 624.2421 683.3072 299.0619 233.1550 520.1370 698.2834 194.9411 297.2447 519.1720 562.4911 177.1642 311.1676 611.1825 628.3977 236.8782 158.1255 593.1619 562.4895 151.1117 311.1702 625.2400 656.4384

318

Table C.2. Confident identifications reported by LipidSearchTM 4.2.21. Reported ions are representative of rat liver and are not the only biomolecules detected.

Table C.3. Lipid classes and IDs confidently detected from flash-frozen and FFPE rat liver slices (10 µm thick).

flash-frozen (FF) FFPE class IDs class IDs WE WE(2:0_16:0) WE WE(4:0_14:0) ChE WE(4:0_16:4) TG TG(4:0_10:4_18:4) Cer ChE(0:0)+H-H2O PC TG(15:0_8:0_11:3) DG Cer(m17:1_17:1) FA PC(18:0_20:4) PS DG(16:2e_18:2) PE FA(20:4) TG PS(4:0_9:0) PA PE(16:0_18:2) PE DG(18:2e_18:2) PS PA(18:0_20:4) BisMePE Cer(m17:0_23:2) PI PE(17:1_18:2) PC Cer(m17:0_24:2) PE(17:1_20:4) FA TG(18:0_6:0_10:3) PC(17:1_18:2) PG DG(18:2e_20:4) PE(18:0_20:4) PI Cer(m17:1_25:1) PE(17:1_22:6) TG(12:0e_6:0_20:4) PC(17:1_20:4) DG(18:0_20:4) PS(18:0e_20:4) PE(16:0_18:2) PC(17:1_22:6) BisMePE(16:0_18:2) PS(18:0_20:4)

319

PE(20:4e_10:4) PI(18:0_20:4) PC(16:0_18:2) PE(18:2_20:4) PE(18:0_20:4) PE(20:0_18:2) PC(18:4_18:0) PC(18:0_18:2) PE(18:0_22:5) BisMePE(18:0_20:4) PS(20:4e_18:0) PC(16:0_22:6) PC(18:0_20:4) PC(17:1_22:6) PC(18:1_22:6) TG(16:0_18:2_18:3) TG(29:1_6:0_20:4) FA(20:5) FA(20:4) FA(22:6) PE(16:0_20:4) PE(18:0_18:2) PE(18:0_22:6) PG(19:1_20:4) PI(18:0_20:4)

320

Figure C.1. Representative MS2 spectra for lipid ion detected both in FF and FFPE tissues (top), only in FFPE (bottom left), and only in FF (bottom right).

321

Appendix D

Supplemental Information for Chapter 5

Table D.1. Parameters for IR-MALDESI ionization source.

x-stage used Newport GTS-70 x-stage typical accuracy ± 0.3 µm x-stage guaranteed accuracy ± 1.0 µm y-stage used Newport LTA-HS y-stage typical accuracy ± 2.2 µm y-stage guaranteed accuracy ± 5.0 µm z-stage used Edmonds optics NT66-508 z-stage accuracy ± 3.0 µm inlet-to-stage distance 6 mm laser-to-inlet distance 5 mm laser-to-ESI emitter tip distance 1 mm

Table D.2. Laser energies and Q-switch delays for depth resolution investigation.

Q-Switch Arbitrary Energy at Target # Delay (µs) Energy (%) (mJ per pulse) 1 680 100 1.20 2 690 89 1.05 3 700 78 1.00 4 710 67 0.90 5 720 56 0.70 6 730 44 0.50 7 740 33 0.30 8 750 22 0.10 (not stable) 9 760 11 could not detect 10 770 0 could not detect

322

Figure D.1. Ion heatmaps of A peaks of three markers: A) starch, B) triethyl citrate, C) omeprazole, and D) their colocalization across 5 layers in the pill trimmed in half

323

Figure D.2. Ion heatmaps of A peaks of three markers: A) starch, B) triethyl citrate, C) omeprazole, and D) their colocalization across 10 layers in the pill trimmed in half.

324

34 Figure D.3. A) The distribution of omeprazole’s S1 peak (m/z 348.1178) on the half pill; B) ion colocalization of A peaks of starch (m/z 163.0601), triethyl citrate (m/z 277.1282) and omeprazole (m/z 346.1220) across 50 layers.

325

Appendix E

Supplemental Information for Chapter 6

Figure E.1. Optical image of spot ablated from ZAP-IT paper at room temperature. Scale bar is 100 µm.

Table E.1. Putative elemental compositions annotated in METASPACE (https://metaspace2020.eu/project/Khodjaniyazova-2019-bones) using Human Metabolome Database for endogenous species.

Healthy Stroke Formula Adduct m/z Formula Adduct m/z C40H80NO8P M+H 734.5694 C40H80NO8P M+H 734.5694 C19H38O2 M+H 299.2944 C37H68O4 M+H 577.5190 C18H36O2 M+H 285.2788 C20H32O2 M+H 305.2475 C37H68O4 M+H 577.5190 C42H82NO8P M+H 760.5850 C20H32O2 M+H 305.2475 C27H44O M+H 385.3465 C27H44O M+H 385.3465 C48H84NO8P M+H 834.6007 C18H34O2 M+H 283.2631 C35H66O4 M+H 551.5033 C30H50 M+H 411.3985 C39H79N2O6P M+H 703.5748 C45H78NO8P M+H 792.5537 C45H78NO8P M+H 792.5537 C10H12N4O5 M+K 307.0439 C37H71O8P M+K 713.4518 C35H66O4 M+H 551.5033 C30H50 M+H 411.3985 C42H82NO8P M+H 760.5850 C35H64O4 M+H 549.4877 C39H79N2O6P M+H 703.5748 C24H50NO7P M+H 496.3397 C22H26O6 M+K 425.1361 C10H17N3O6S M+H 308.0910 C18H30O M+H 263.2369 C26H33NO6 M+H 456.2380 C18H34O4 M+H 315.2529 C22H32O2 M+H 329.2475 C24H50NO7P M+H 496.3397 C23H45NO4 M+H 400.3421 C29H46O3 M+Na 465.3339 C46H80NO8P M+H 806.5694

326

C48H84NO8P M+H 834.6007 C44H80NO8P M+H 782.5694 C35H64O4 M+H 549.4877 C46H84NO8P M+H 810.6007 C22H32O2 M+H 329.2475 C43H74NO8P M+H 764.5224 C10H17N3O6S M+H 308.0910 C18H13NO4 M+H 308.0917 C15H10N2O2 M+H 251.0815 C37H74NO8P M+H 692.5224 C41H83N2O6P M+H 731.6061 C27H44O2 M+H 401.3414 C22H45NO M+H 340.3574 C37H66O4 M+H 575.5033 C43H74NO8P M+H 764.5224 C27H42O M+H 383.3308 C20H30O2 M+H 303.2318 C42H84NO7P M+H 746.6058 C37H66O4 M+H 575.5033 C29H48O3 M+H 445.3676 C46H80NO8P M+H 806.5694 C25H47NO4 M+H 426.3577 C26H54NO7P M+H 524.3710 C42H80NO8P M+H 758.5694 C18H13NO4 M+H 308.0917 C26H54NO7P M+H 524.3710 C42H84NO7P M+H 746.6058 C22H34O2 M+H 331.2631 C37H74NO8P M+H 692.5224 C38H76NO8P M+H 706.5381 C46H84NO8P M+H 810.6007 C20H30O2 M+H 303.2318 C27H44O2 M+H 401.3414 C40H78NO8P M+H 732.5537 C44H80NO8P M+H 782.5694 C43H78NO8P M+H 768.5537 C29H57NO4 M+H 484.4360 C41H83N2O6P M+H 731.6061 C22H34O2 M+H 331.2631 C44H82NO7P M+H 768.5901 C27H46O2 M+H 403.3570 C27H46O2 M+H 403.3570 C27H42O M+H 383.3308 C44H84NO8P M+H 786.6007 C21H40O3 M+H 341.3050 C47H93N2O6P M+H 813.6844 C16H12N2O2 M+H 265.0971 C18H39N M+H 270.3155 C42H80NO8P M+H 758.5694 C35H62O4 M+H 547.4720 C23H45NO4 M+H 400.3421 C18H35NO M+H 282.2791 C26H33NO6 M+H 456.2380 C12H17N3O4 M+H 268.1291 C37H64O4 M+H 573.4877 C41H74NO7P M+H 724.5275 C35H62O4 M+H 547.4720 C37H64O4 M+H 573.4877 C44H82NO7P M+H 768.5901 C42H82NO7P M+H 744.5901 C43H78NO8P M+H 768.5537 C43H74NO7P M+H 748.5275 C18H35NO M+H 282.2791 C42H78NO8P M+H 756.5537 C44H84NO8P M+H 786.6007 C39H74NO8P M+H 716.5224 C40H78NO8P M+H 732.5537 C46H84NO7P M+H 794.6058 C47H93N2O6P M+H 813.6844 C46H82NO8P M+H 808.5850 C38H76NO8P M+H 706.5381 C22H35NO2 M+H 346.2740 C20H35NO2 M+H 322.2740 C43H78NO7P M+H 752.5588 C20H30O M+H 287.2369 C41H78NO8P M+H 744.5537 C43H74NO7P M+H 748.5275 C44H78NO8P M+H 780.5537 C29H48O3 M+H 445.3676 C46H78NO8P M+H 804.5537 C42H78NO8P M+H 756.5537 C48H82NO8P M+H 832.5850

327

C42H82NO7P M+H 744.5901 C21H39O6P M+H 419.2557 C34H67NO3 M+H 538.5193 C12H20N4O3 M+H 269.1608 C46H82NO8P M+H 808.5850 C36H71NO3 M+H 566.5506 C21H39O6P M+H 419.2557 C45H69O8P M+H 769.4802 C34H65NO3 M+H 536.5037 C21H40O3 M+H 341.3050 C21H42O4 M+H 359.3155 C26H52NO6P M+H 506.3605 C21H36O3 M+H 337.2737 C39H76NO8P M+H 718.5381 C44H78NO8P M+H 780.5537 C22H36O2 M+H 333.2788 C39H74NO8P M+H 716.5224 C43H76NO8P M+H 766.5381 C44H82NO8P M+H 784.5850 C44H76NO8P M+H 778.5381 C46H78NO8P M+H 804.5537 C10H14N5O7P M+H 348.0703 C48H82NO8P M+H 832.5850 C23H48NO7P M+H 482.3241 C24H40O4 M+H 393.2999 C44H82NO8P M+H 784.5850 C39H76NO8P M+H 718.5381 C39H69O8P M+H 697.4802 C46H84NO7P M+H 794.6058 C55H98O6 M+H 855.7436 C21H40O4 M+H 357.2999 C41H80NO8P M+H 746.5694 C57H100O6 M+H 881.7592 C45H76NO8P M+H 790.5381 C41H74NO7P M+H 724.5275 C45H91N2O6P M+H 787.6687 C43H78NO7P M+H 752.5588 C40H80NO7P M+H 718.5745 C41H78NO8P M+H 744.5537 C25H49NO4 M+H 428.3734 C39H69O8P M+H 697.4802 C20H35NO2 M+H 322.2740 C55H98O6 M+H 855.7436 C42H84NO8P M+H 762.6007 C12H17N3O4 M+H 268.1291 C34H65NO3 M+H 536.5037 C22H35NO2 M+H 346.2740 C41H74NO8P M+H 740.5224 C48H80NO8P M+H 830.5694 C13H24N4O3 M+H 285.1921 C55H100O6 M+H 857.7592 C46H86NO7P M+H 796.6214 C44H76NO8P M+H 778.5381 C26H46O6 M+H 455.3367 C39H70O5 M+H 619.5296 C30H58O4S M+H 515.4128 C53H96O6 M+H 829.7279 C11H18N4O3 M+H 255.1451 C40H80NO7P M+H 718.5745 C57H100O6 M+H 881.7592 C41H71O8P M+H 723.4959 C40H76NO8P M+H 730.5381 C43H76NO8P M+H 766.5381 C41H78NO7P M+H 728.5588 C45H76NO8P M+H 790.5381 C44H86NO7P M+H 772.6214 C59H102O6 M+H 907.7749 C27H44O3 M+H 417.3363 C22H36O2 M+H 333.2788 C27H45O5 M+H 450.3339 C46H80NO7P M+H 790.5745 C55H96O6 M+H 853.7279 C46H86NO7P M+H 796.6214 C55H100O6 M+H 857.7592 C26H52NO6P M+H 506.3605 C48H80NO8P M+H 830.5694 C45H69O8P M+H 769.4802 C27H42O3 M+H 415.3206 C27H45O5 M+H 450.3339 C27H46O M+H 387.3621 C45H91N2O6P M+H 787.6687 C41H71O8P M+H 723.4959

328

C26H48O5 M+H 441.3574 C42H76NO8P M+H 754.5381 C36H73NO3 M+H 568.5663 C53H92O7 M+H 841.6915 C50H82NO8P M+H 856.5850 C47H95N2O6P M+H 815.7000 C22H36O4 M+H 365.2686 C53H96O6 M+H 829.7279 C12H20N4O3 M+H 269.1608 C48H78NO8P M+H 828.5537 C47H82NO8P M+H 820.5850 C57H98O6 M+H 879.7436 C26H50O5 M+H 443.3731 C47H82NO8P M+H 820.5850 C27H44O3 M+H 417.3363 C29H50O2 M+H 431.3883 C26H42O M+H 371.3308 C18H33NO M+H 280.2635 C44H86NO8P M+H 788.6163 C44H80NO7P M+H 766.5745 C55H96O6 M+H 853.7279 C55H94O6 M+H 851.7123 C42H84NO8P M+H 762.6007 C21H41NO4 M+H 372.3108 C27H53NO4 M+H 456.4047 C43H76NO7P M+H 750.5432 C10H14N5O7P M+H 348.0703 C44H86NO8P M+H 788.6163 C25H47NO4 M+H 426.3577 C44H85NO11S M+H 836.5916 C47H76NO8P M+H 814.5381 C21H40O4 M+H 357.2999 C44H84NO6P M+H 754.6109 C39H78NO8P M+H 720.5537 C46H82NO7P M+H 792.5901 C44H84NO6P M+H 754.6109 C41H74NO8P M+H 740.5224 C59H102O6 M+H 907.7749 C36H71NO3 M+H 566.5506 C21H36O3 M+H 337.2737 C47H73O8P M+H 797.5115 C34H67NO3 M+H 538.5193 C11H18N4O3 M+H 255.1451 C47H80NO8P M+H 818.5694 C39H68O5 M+H 617.5139 C28H48O5 M+H 465.3574 C47H95N2O6P M+H 815.7000 C37H72NO8P M+H 690.5068 C30H58O4S M+H 515.4128 C45H78NO7P M+H 776.5588 C53H92O7 M+H 841.6915 C46H80NO7P M+H 790.5745 C57H102O6 M+H 883.7749 C57H96O6 M+H 877.7279 C44H80NO7P M+H 766.5745 C47H78NO8P M+H 816.5537 C25H49NO4 M+H 428.3734 C44H84NO7P M+H 770.6058 C27H46O M+H 387.3621 C13H17N3O4 M+H 280.1291 C41H80NO8P M+H 746.5694 C22H39NO2 M+H 350.3053 C20H32N6O12S2 M+H 613.1592 C43H69O8P M+H 745.4802 C44H86NO7P M+H 772.6214 C13H20N2O3 M+H 253.1546 C27H42O3 M+H 415.3206 C41H76NO8P M+H 742.5381 C42H76NO8P M+H 754.5381 C57H102O6 M+H 883.7749 C57H98O6 M+H 879.7436 C43H80NO8P M+H 770.5694 C40H76NO8P M+H 730.5381 C16H21N3O3S M+H 336.1376 C43H76NO7P M+H 750.5432 C41H69O8P M+H 721.4802 C26H52NO7P M+H 522.3554 C59H98O6 M+H 903.7436 C19H38O4 M+H 331.2842 C50H82NO8P M+H 856.5850 C45H78NO7P M+H 776.5588 C8H20NO6P M+H 258.1101

329

C42H80NO7P M+H 742.5745 C46H82NO7P M+H 792.5901 C59H100O6 M+H 905.7592 C26H40O M+H 369.3152 C43H82NO8P M+H 772.5850 C14H19N3O4 M+H 294.1448 C47H80NO8P M+H 818.5694 C39H70O5 M+H 619.5296 C48H78NO8P M+H 828.5537 C42H81NO3 M+H 648.6289 C42H81NO3 M+H 648.6289 C43H82NO8P M+H 772.5850 C43H69O8P M+H 745.4802 C26H42O M+H 371.3308 C16H22O5 M+H 295.1540 C39H68O5 M+H 617.5139 C43H73O8P M+H 749.5115 C29H48O2 M+H 429.3727 C41H69O8P M+H 721.4802 C45H80NO8P M+H 794.5694 C44H84NO7P M+H 770.6058 C11H16N4O4 M+H 269.1244 C25H42O4 M+H 407.3155 C11H16N4O3 M+H 253.1295 C41H76NO8P M+H 742.5381 C47H73O8P M+H 797.5115 C28H48O4 M+H 449.3625 C27H40O2 M+H 397.3101 C53H94O6 M+H 827.7123 C15H19N3O2 M+H 274.1550 C41H78NO7P M+H 728.5588 C10H13N4O8P M+H 349.0543 C43H80NO8P M+H 770.5694 C15H19N3O3 M+H 290.1499 C26H46O6 M+H 455.3367 C16H18N3O5 M+H 333.1319 C38H77NO3 M+H 596.5976 C43H71O8P M+H 747.4959 C27H40O2 M+H 397.3101 C25H36O3 M+H 385.2737 C22H39NO2 M+H 350.3053 C14H20N2O3 M+H 265.1546 C39H78NO7P M+H 704.5588 C14H19N3O2 M+H 262.1550 C22H37NO2 M+H 348.2897 C39H78NO7P M+H 704.5588 C13H24N4O3 M+H 285.1921 C42H80NO7P M+H 742.5745 C12H16O6S M+H 289.0740 C15H23N3O2 M+H 278.1863 C47H74O2 M+H 671.5761 C45H73O8P M+H 773.5115 C59H98O6 M+H 903.7436 C25H47NO5 M+H 442.3527 C57H96O6 M+H 877.7279 C23H45NO5 M+H 416.3370 C23H38O4 M+H 379.2842 C37H69O7P M+H 657.4853 C24H37NO2 M+H 372.2897 C46H86NO8P M+H 812.6163 C26H47NO M+H 390.3730 C26H47NO M+H 390.3730 C26H40O M+H 369.3152 C47H76NO8P M+H 814.5381 C46H86NO8P M+H 812.6163 C18H19NO7 M+H 362.1234 C15H12N2O2 M+H 253.0971 C38H74NO8P M+H 704.5224 C50H80NO8P M+H 854.5694 C22H37NO2 M+H 348.2897 C38H78NO7P M+H 692.5588 C49H100N2O6P M+H 844.7391 C37H72NO8P M+H 690.5068 C37H66O5 M+H 591.4983 C49H100N2O6P M+H 844.7391 C38H78NO7P M+H 692.5588 C54H82O4 M+H 795.6285 C14H18N2O3 M+H 263.1390 C55H94O6 M+H 851.7123 C23H46NO7P M+H 480.3084 C11H16N4O4 M+H 269.1244 C10H13N5O3 M+H 252.1091

330

C21H41NO4 M+H 372.3108 C21H22O7 M+H 387.1438 C11H12BrN5O M+H 310.0298 C57H104O6 M+H 885.7905 C10H13N4O8P M+H 349.0543 C45H82NO8P M+H 796.5850 C25H45NO4 M+H 424.3421 C53H94O6 M+H 827.7123 C45H71O8P M+H 771.4959 C19H40O3 M+H 317.3050 C41H68O5 M+H 641.5139 C19H22O7 M+H 363.1438 C11H16N4O3 M+H 253.1295 C32H50O7 M+H 547.3629 C43H71O8P M+H 747.4959 C13H18N2O3 M+H 251.1390 C39H76NO7P M+H 702.5432 C21H23NO7 M+H 402.1547 C38H70O4 M+H 591.5346 C42H86NO7P M+H 748.6214 C39H78NO8P M+H 720.5537 C19H20O7 M+H 361.1281 C20H24O7 M+H 377.1594 C26H52NO7P M+H 522.3554 C44H85NO11S M+H 836.5916 C43H73O8P M+H 749.5115 C16H21N3O3S M+H 336.1376 C23H31NO7 M+H 434.2173 C45H80NO8P M+H 794.5694 C48H86NO8P M+H 836.6163 C20H39NO2 M+H 326.3053 C22H39NO M+H 334.3104 C41H70O5 M+H 643.5296 C41H76NO7P M+H 726.5432 C26H56NO6P M+H 510.3918 C23H43NO4 M+H 398.3264 C23H46NO7P M+H 480.3084 C23H29NO7 M+H 432.2016 C29H48O2 M+H 429.3727 C24H37NO2 M+H 372.2897 C16H12O9 M+H 349.0554 C14H17N3O3 M+H 276.1342 C45H82NO8P M+H 796.5850 C20H24O7 M+H 377.1594 C18H18O6 M+H 331.1176 C20H32N6O12S2 M+H 613.1592 C26H50NO7P M+H 520.3397 C14H22N2O3 M+H 267.1703 C41H76NO7P M+H 726.5432 C21H24O7 M+H 389.1594 C35H52O4 M+H 537.3938 C39H81N2O6P M+H 705.5905 C13H20N2O3 M+H 253.1546 C45H71O8P M+H 771.4959 C26H54NO6P M+H 508.3761 C23H27NO9 M+H 462.1758 C45H73O8P M+H 773.5115 C39H76NO7P M+H 702.5432 C15H10O4 M+H 255.0651 C18H20O7 M+H 349.1281 C45H67O8P M+H 767.4646 C20H23NO6 M+H 374.1598 C29H50O2 M+H 431.3883 C43H80NO7P M+H 754.5745 C25H36O3 M+H 385.2737 C18H18O7 M+H 347.1125 C23H48NO7P M+H 482.3241 C14H22N2O2 M+H 251.1754 C39H67O8P M+H 695.4646 C15H14O7 M+H 307.0812 C45H76NO7P M+H 774.5432 C45H76NO7P M+H 774.5432 C37H69O7P M+H 657.4853 C26H50NO7P M+H 520.3397 C42H86NO7P M+H 748.6214 C50H80NO8P M+H 854.5694 C43H87N2O6P M+H 759.6374 C16H12O9 M+H 349.0554 C27H46O3 M+H 419.3519 C44H74NO8P M+H 776.5224 C42H83NO3 M+H 650.6445 C15H10O4 M+H 255.0651

331

C10H13N5O3 M+H 252.1091 C54H82O4 M+H 795.6285 C12H5F6NO2 M+H 310.0297 C18H20O6 M+H 333.1332 C14H30N4O2 M+H 287.2441 C17H18O8 M+H 351.1074 C51H90O7 M+H 815.6759 C22H28O7 M+H 405.1907 C39H81N2O6P M+H 705.5905 C19H21NO6 M+H 360.1441 C19H20O7 M+H 361.1281 C42H83NO3 M+H 650.6445 C19H40O3 M+H 317.3050 C16H12O4 M+H 269.0808 C46H78NO10P M+H 836.5436 C20H18O8 M+H 387.1074 C15H10N2O3 M+H 267.0764 C10H16N4O4 M+H 257.1244 C21H23NO7 M+H 402.1547 C45H82NO7P M+H 780.5901 C13H17N3O4 M+H 280.1291 C18H18O8 M+H 363.1074 C45H74NO8P M+H 788.5224 C41H68O5 M+H 641.5139 C18H20O7 M+H 349.1281 C20H20O7 M+H 373.1281 C49H82NO8P M+H 844.5850 C22H25NO7 M+H 416.1703 C41H80NO7P M+H 730.5745 C45H80NO7P M+H 778.5745 C43H80NO7P M+H 754.5745 C46H76NO8P M+H 802.5381 C11H15N5O3S M+H 298.0968 C23H39NO M+H 346.3104 C48H86NO8P M+H 836.6163 C59H100O6 M+H 905.7592 C37H66O5 M+H 591.4983 C41H80NO7P M+H 730.5745 C21H44O3 M+H 345.3363 C16H20N2O M+H 257.1648 C48H84NO7P M+H 818.6058 C41H82NO7P M+H 732.5901 C8H20NO6P M+H 258.1101 C22H30O2 M+H 327.2318 C16H16O7 M+H 321.0968 C18H20FNO3S M+H 350.1220 C17H12O4 M+H 281.0808 C43H87N2O6P M+H 759.6374 C13H18N2O3 M+H 251.1390 C26H54NO6P M+H 508.3761 C27H40O3 M+H 413.3050 C23H28O7 M+H 417.1907 C20H37NO2 M+H 324.2897 C34H69NO3 M+H 540.5350 C27H42O4 M+H 431.3155 C42H81NO11S M+H 808.5603 C22H25NO7 M+H 416.1703 C48H86NO7P M+H 820.6214 C25H47NO5 M+H 442.3527 C39H74NO7P M+H 700.5275 C52H80NO8P M+H 878.5694 C41H72NO8P M+H 738.5068 C37H74O13P2 M+H 789.4677 C17H14O4 M+H 283.0964 C45H72NO8P M+H 786.5068 C27H45NO M+H 400.3574 C23H39NO M+H 346.3104 C32H45NO4 M+H 508.3421 C53H92O6 M+H 825.6966 C25H50NO7P M+H 508.3397 C39H74NO7P M+H 700.5275 C12H16N2O4 M+H 253.1182 C53H90O7 M+H 839.6759 C14H17N2O5 M+H 294.1210 C41H82NO8P M+H 748.5850 C15H19N3O4 M+H 306.1448 C27H36N2O5 M+H 469.2697 C19H20O8 M+H 377.1231 C14H12O6S M+H 309.0427 C17H18O6 M+H 319.1176 C45H80NO7P M+H 778.5745 C27H42O4 M+H 431.3155

332

C36H68O4 M+H 565.5190 C14H17N3O2 M+H 260.1393 C27H45NO2 M+H 416.3523 C53H92O6 M+H 825.6966 C18H19NO7 M+H 362.1234 C17H18O7 M+H 335.1125 C23H43NO5 M+H 414.3214 C19H24O7 M+H 365.1594 C49H84NO8P M+H 846.6007 C43H82NO7P M+H 756.5901 C14H19N3O4 M+H 294.1448 C10H13N5O4 M+H 268.1040 C30H52O4 M+H 477.3938 C37H70O12S M+H 739.4660 C18H20O6 M+H 333.1332 C60H94O6 M+H 911.7123 C32H45NO4 M+H 508.3421 C40H74NO8P M+H 728.5224 C24H48NO7P M+H 494.3241 C41H70O5 M+H 643.5296 C41H82NO7P M+H 732.5901 C20H39NO5 M+H 374.2901 C27H45NO M+H 400.3574 C11H15N5O3S M+H 298.0968 C57H104O6 M+H 885.7905 C25H48NO7P M+H 506.3241 C10H16N4O4 M+H 257.1244 C39H66O5 M+H 615.4983 C39H66O5 M+H 615.4983 C25H38O4 M+H 403.2842 C47H93O8P M+H 817.6680 C21H23NO9 M+H 434.1445 C19H22O7 M+H 363.1438 C40H78NO7P M+H 716.5588 C57H94O6 M+H 875.7123 C53H90O7 M+H 839.6759 C43H82NO7P M+H 756.5901 C52H80NO8P M+H 878.5694 C28H52NO7P M+H 546.3554 C44H86NO6P M+H 756.6265 C22H39NO M+H 334.3104 C14H30N4O2 M+H 287.2441 C42H81NO11S M+H 808.5603 C25H32N2O7 M+H 473.2282 C23H43NO4 M+H 398.3264 C24H48NO7P M+H 494.3241 C20H22O7 M+H 375.1438 C34H39N3O12S M+H 714.2327 C48H86NO7P M+H 820.6214 C15H23N3O3 M+H 294.1812 C34H69NO3 M+H 540.5350 C21H39NO4 M+H 370.2951 C20H26O7 M+H 379.1751 C7H14N2O6S M+H 255.0645 C45H82NO7P M+H 780.5901 C19H19NO6 M+H 358.1285 C14H20N2O3 M+H 265.1546 C29H38O4 M+H 451.2842 C21H26O7 M+H 391.1751 C46H93N2O6P M+H 801.6844 C40H74NO8P M+H 728.5224 C24H32O7 M+H 433.2220 C40H75NO8 M+H 698.5565 C23H29NO8 M+H 448.1966 C14H17N2O5 M+H 294.1210 C45H72NO8P M+H 786.5068 C13H25O7P M+H 325.1410 C48H84NO7P M+H 818.6058 C44H78NO10P M+H 812.5436 C48H77O10P M+H 845.5327 C44H86NO6P M+H 756.6265 C49H82NO8P M+H 844.5850 C19H24O7 M+H 365.1594 C14H12O6S M+H 309.0427 C37H64O5 M+H 589.4826 C43H72NO8P M+H 762.5068 C50H84NO8P M+H 858.6007 C17H25N5O4 M+H 364.1979 C24H47NO4 M+H 414.3577 C23H40NO7P M+H 474.2615 C10H14N5O6P M+H 332.0754 C20H37NO2 M+H 324.2897

333

C22H22O11 M+H 463.1234 C20H22O7 M+H 375.1438 C23H27NO9 M+H 462.1758 C10H14N5O6P M+H 332.0754 C15H12O4 M+H 257.0808 C43H84NO8P M+H 774.6007 C17H18O7 M+H 335.1125 C18H18O6 M+H 331.1176 C16H16O6 M+H 305.1019 C49H84NO8P M+H 846.6007 C34H64O4 M+H 537.4877 C20H26O7 M+H 379.1751 C40H78NO7P M+H 716.5588 C13H26N4O2 M+H 271.2128 C41H66O5 M+H 639.4983 C25H43NO4 M+H 422.3264 C43H72O5 M+H 669.5452 C21H26O7 M+H 391.1751 C39H82N2O6P M+H 706.5983 C23H30O7 M+H 419.2064 C14H17NO7 M+H 312.1077 C46H78NO10P M+H 836.5436 C43H84NO8P M+H 774.6007 C24H42O12 M+H 523.2749 C50H78NO8P M+H 852.5537 C37H40N2O6 M+H 609.2959 C37H40N2O6 M+H 609.2959 C23H44NO7P M+H 478.2928 C22H28F2O5 M+H 411.1977 C45H74NO8P M+H 788.5224 C7H14N2O6S M+H 255.0645 C16H16O6 M+H 305.1019 C20H32O4 M+H 337.2373 C17H24N4O3 M+H 333.1921 C37H70O12S M+H 739.4660 C55H102O6 M+H 859.7749 C23H44NO7P M+H 478.2928 C24H40O4 M+H 393.2999 C15H23N3O2 M+H 278.1863 C27H40O3 M+H 413.3050 C53H98O6 M+H 831.7436 C53H98O6 M+H 831.7436 C9H16N2O6S M+H 281.0801 C19H37O6P M+H 393.2400 C16H12O4 M+H 269.0808 C16H26O8 M+H 347.1700 C48H90NO7P M+H 824.6527 C39H67O8P M+H 695.4646 C10H13N5O4 M+H 268.1040 C21H27NO6 M+H 390.1911 C25H38O4 M+H 403.2842 C41H74O12S M+H 791.4973 C14H19N3O2 M+H 262.1550 C42H79NO11S M+H 806.5446 C25H48NO7P M+H 506.3241 C13H18N2O4 M+H 267.1339 C50H86NO8P M+H 860.6163 C35H53O8 M+H 602.3813 C10H8O7S M+H 273.0063 C48H82NO7P M+H 816.5901 C23H31NO7 M+H 434.2173 C37H64O5 M+H 589.4826 C47H78NO8P M+H 816.5537 C21H22O8 M+H 403.1387 C18H14O4 M+H 295.0964 C11H21N5O5 M+H 304.1615 C38H74NO8P M+H 704.5224 C50H86NO8P M+H 860.6163 C21H22O7 M+H 387.1438 C9H14N2O7 M+H 263.0873 C37H70O5 M+H 595.5296 C43H84NO7P M+H 758.6058 C55H102O6 M+H 859.7749 C16H18O6 M+H 307.1176 C17H14O4 M+H 283.0964 C35H70NO8P M+H 664.4911 C23H29NO8 M+H 448.1966 C30H50NO7P M+H 568.3397 C37H68O5 M+H 593.5139 C18H22N2O M+H 283.1805 C17H26N4O2 M+H 319.2128 C14H17N3O4 M+H 292.1291

334

C18H18O7 M+H 347.1125 C39H40O15 M+H 749.2440 C47H84NO8P M+H 822.6007 C27H40O4 M+H 429.2999 C15H19N3O3 M+H 290.1499 C24H47NO4 M+H 414.3577 C20H21NO7 M+H 388.1390 C17H12O4 M+H 281.0808 C14H18N5O11P M+H 464.0813 C18H18FNO2S M+H 332.1115 C16H18N3O5 M+H 333.1319 C41H82NO8P M+H 748.5850 C21H43O6P M+H 423.2870 C50H84NO8P M+H 858.6007 C40H60O4 M+H 605.4564 C19H24O6 M+H 349.1645 C46H81O10P M+H 825.5640 C14H17NO7 M+H 312.1077 C16H18O6 M+H 307.1176 C41H82O13P2 M+H 845.5303 C13H22N4O8S2 M+H 427.0951 C18H14O4 M+H 295.0964 C42H78NO7P M+H 740.5588 C20H22O8 M+H 391.1387 C59H96O6 M+H 901.7279 C18H22O8 M+H 367.1387 C43H72NO8P M+H 762.5068 C14H22N2O3 M+H 267.1703 C26H45NO M+H 388.3574 C42H76NO7P M+H 738.5432 C23H45NO5 M+H 416.3370 C26H32O3 M+H 393.2424 C11H21N5O3 M+H 272.1717 C55H92O6 M+H 849.6966 C17H26O4 M+H 295.1903 C46H93N2O6P M+H 801.6844 C36H71O8P M+H 663.4959 C36H72NO6P M+H 646.5170 C24H32O7 M+H 433.2220 C21H24O7 M+H 389.1594 C46H88NO8P M+H 814.6320 C43H66O5 M+H 663.4983 C43H85NO8 M+H 744.6348 C16H10O5 M+H 283.0601 C8H16NO3PS2 M+H 270.0382 C16H13NO4 M+H 284.0917 C26H32O4 M+H 409.2373 C29H38O4 M+H 451.2842 C15H19N3O2 M+H 274.1550 C21H20O11 M+H 449.1078 C16H24N3O8 M+H 387.1636 C22H30O2 M+H 327.2318 C20H18O7S M+H 403.0846

335

C25H50NO7P M+H 508.3397 C15H31O7P M+H 355.1880 C43H70O5 M+H 667.5296 C48H77O10P M+H 845.5327 C21H37O6P M+H 417.2400 C14H18N2O3 M+H 263.1390 C38H75NO3 M+H 594.5819 C26H48NO7P M+H 518.3241 C44H78NO7P M+H 764.5588 C48H76NO8P M+H 826.5381 C16H20N2O M+H 257.1648 C48H82NO7P M+H 816.5901 C41H76NO10P M+H 774.5279 C22H28O7 M+H 405.1907 C47H86O6 M+H 747.6497 C19H22O6 M+H 347.1489 C45H84NO8P M+H 798.6007 C27H38O2 M+H 395.2944 C16H14O4 M+H 271.0964 C33H64O3 M+H 509.4928 C21H41O6P M+H 421.2713 C9H14N2O7 M+H 263.0873 C26H42O3 M+H 403.3206 C44H88NO7P M+H 774.6371 C24H41NO2 M+H 376.3210

Table E.2. Putative elemental compositions annotated in METASPACE (https://metaspace2020.eu/project/Khodjaniyazova-2019-bones) using LipidMaps database.

Healthy Stroke Formula Adduct m/z Formula Adduct m/z C27H44 M+H 369.3515 C27H44 M+H 369.3515 C26H50O4 M+H 427.3781 C40H80NO8P M+H 734.5694 C26H50O4 M+Na 449.3601 C37H68O4 M+H 577.5190 C40H80NO8P M+H 734.5694 C20H32O2 M+H 305.2475 C19H38O2 M+H 299.2944 C40H82NO7P M+H 720.5901 C18H36O2 M+H 285.2788 C42H82NO8P M+H 760.5850 C37H68O4 M+H 577.5190 C27H44O M+H 385.3465 C20H32O2 M+H 305.2475 C26H50O4 M+H 427.3781 C18H32O M+H 265.2526 C48H84NO8P M+H 834.6007

336

C40H82NO7P M+H 720.5901 C35H66O4 M+H 551.5033 C27H44O M+H 385.3465 C39H79N2O6P M+H 703.5748 C18H34O2 M+H 283.2631 C45H78NO8P M+H 792.5537 C30H50 M+H 411.3985 C39H70O4 M+H 603.5346 C26H50O4 M+K 465.3340 C34H67NO2 M+H 522.5244 C39H70O4 M+H 603.5346 C23H36O4 M+Na 399.2505 C45H78NO8P M+H 792.5537 C37H71O8P M+K 713.4518 C35H66O4 M+H 551.5033 C30H50 M+H 411.3985 C42H82NO8P M+H 760.5850 C24H50NO7P M+H 496.3397 C39H79N2O6P M+H 703.5748 C24H48NO6P M+H 478.3292 C22H26O6 M+K 425.1361 C22H32O2 M+H 329.2475 C18H30O M+H 263.2369 C23H45NO4 M+H 400.3421 C19H36O3 M+H 313.2737 C46H80NO8P M+H 806.5694 C25H22O5 M+Na 425.1359 C44H80NO8P M+H 782.5694 C18H34O4 M+H 315.2529 C46H84NO8P M+H 810.6007 C24H50NO7P M+H 496.3397 C43H74NO8P M+H 764.5224 C29H46O3 M+Na 465.3339 C37H74NO8P M+H 692.5224 C48H84NO8P M+H 834.6007 C27H44O2 M+H 401.3414 C43H71O8P M+Na 769.4778 C42H81NO2 M+H 632.6340 C22H32O2 M+H 329.2475 C37H66O4 M+H 575.5033 C29H47NO4 M+Na 496.3397 C27H42O M+H 383.3308 C41H83N2O6P M+H 731.6061 C42H84NO7P M+H 746.6058 C24H48NO6P M+H 478.3292 C29H48O3 M+H 445.3676 C34H67NO2 M+H 522.5244 C25H47NO4 M+H 426.3577 C22H45NO M+H 340.3574 C30H48 M+H 409.3828 C43H74NO8P M+H 764.5224 C42H80NO8P M+H 758.5694 C37H71O8P M+K 713.4518 C26H54NO7P M+H 524.3710 C20H30O2 M+H 303.2318 C22H34O2 M+H 331.2631 C37H66O4 M+H 575.5033 C38H76NO8P M+H 706.5381 C46H80NO8P M+H 806.5694 C20H30O2 M+H 303.2318 C26H54NO7P M+H 524.3710 C40H78NO8P M+H 732.5537 C42H84NO7P M+H 746.6058 C43H78NO8P M+H 768.5537 C37H74NO8P M+H 692.5224 C41H83N2O6P M+H 731.6061 C46H84NO8P M+H 810.6007 C44H82NO7P M+H 768.5901 C27H44O2 M+H 401.3414 C27H46O2 M+H 403.3570 C44H80NO8P M+H 782.5694 C44H84NO8P M+H 786.6007 C29H57NO4 M+H 484.4360 C27H42O2 M+H 399.3257 C22H34O2 M+H 331.2631 C47H93N2O6P M+H 813.6844 C28H44F2O3 M+H 467.3331 C30H52O5 M+H 493.3887 C27H46O2 M+H 403.3570 C35H62O4 M+H 547.4720 C27H42O M+H 383.3308 C18H35NO M+H 282.2791

337

C21H40O3 M+H 341.3050 C41H74NO7P M+H 724.5275 C42H81NO2 M+H 632.6340 C42H82NO7P M+H 744.5901 C42H80NO8P M+H 758.5694 C43H74NO7P M+H 748.5275 C23H45NO4 M+H 400.3421 C42H78NO8P M+H 756.5537 C35H62O4 M+H 547.4720 C39H74NO8P M+H 716.5224 C44H82NO7P M+H 768.5901 C46H84NO7P M+H 794.6058 C43H78NO8P M+H 768.5537 C46H82NO8P M+H 808.5850 C18H35NO M+H 282.2791 C22H35NO2 M+H 346.2740 C44H84NO8P M+H 786.6007 C43H70O4 M+H 651.5346 C16H27F3O2 M+H 309.2036 C43H78NO7P M+H 752.5588 C40H78NO8P M+H 732.5537 C41H78NO8P M+H 744.5537 C47H93N2O6P M+H 813.6844 C44H78NO8P M+H 780.5537 C38H76NO8P M+H 706.5381 C46H78NO8P M+H 804.5537 C20H35NO2 M+H 322.2740 C48H82NO8P M+H 832.5850 C20H30O M+H 287.2369 C25H46NO6P M+H 488.3135 C43H74NO7P M+H 748.5275 C21H39O6P M+H 419.2557 C29H48O3 M+H 445.3676 C36H71NO3 M+H 566.5506 C42H78NO8P M+H 756.5537 C45H69O8P M+H 769.4802 C42H82NO7P M+H 744.5901 C21H40O3 M+H 341.3050 C34H67NO3 M+H 538.5193 C26H52NO6P M+H 506.3605 C27H42O2 M+H 399.3257 C45H74NO7P M+H 772.5275 C46H82NO8P M+H 808.5850 C39H76NO8P M+H 718.5381 C43H70O4 M+H 651.5346 C22H36O2 M+H 333.2788 C21H39O6P M+H 419.2557 C47H91N2O6P M+H 811.6687 C34H65NO3 M+H 536.5037 C43H76NO8P M+H 766.5381 C21H42O4 M+H 359.3155 C27H51NO4 M+H 454.3890 C44H78NO8P M+H 780.5537 C44H76NO8P M+H 778.5381 C39H74NO8P M+H 716.5224 C23H48NO7P M+H 482.3241 C44H82NO8P M+H 784.5850 C39H71O7P M+H 683.5010 C46H78NO8P M+H 804.5537 C28H50O5 M+H 467.3731 C48H82NO8P M+H 832.5850 C25H45NO2 M+H 392.3523 C24H40O4 M+H 393.2999 C44H82NO8P M+H 784.5850 C39H76NO8P M+H 718.5381 C39H69O8P M+H 697.4802 C46H84NO7P M+H 794.6058 C55H98O6 M+H 855.7436 C21H40O4 M+H 357.2999 C41H80NO8P M+H 746.5694 C57H100O6 M+H 881.7592 C45H76NO8P M+H 790.5381 C47H91N2O6P M+H 811.6687 C45H91N2O6P M+H 787.6687 C41H74NO7P M+H 724.5275 C40H80NO7P M+H 718.5745 C43H78NO7P M+H 752.5588 C25H49NO4 M+H 428.3734 C45H74NO7P M+H 772.5275 C20H35NO2 M+H 322.2740 C30H48 M+H 409.3828 C42H84NO8P M+H 762.6007

338

C41H78NO8P M+H 744.5537 C34H65NO3 M+H 536.5037 C39H69O8P M+H 697.4802 C27H49NO2 M+H 420.3836 C55H98O6 M+H 855.7436 C41H74NO8P M+H 740.5224 C25H46NO6P M+H 488.3135 C46H86NO7P M+H 796.6214 C24H52NO6P M+H 482.3605 C26H46O6 M+H 455.3367 C22H35NO2 M+H 346.2740 C57H100O6 M+H 881.7592 C48H80NO8P M+H 830.5694 C40H76NO8P M+H 730.5381 C55H100O6 M+H 857.7592 C41H78NO7P M+H 728.5588 C44H76NO8P M+H 778.5381 C44H86NO7P M+H 772.6214 C39H70O5 M+H 619.5296 C27H44O3 M+H 417.3363 C53H96O6 M+H 829.7279 C55H96O6 M+H 853.7279 C40H80NO7P M+H 718.5745 C55H100O6 M+H 857.7592 C41H71O8P M+H 723.4959 C48H80NO8P M+H 830.5694 C43H76NO8P M+H 766.5381 C27H42O3 M+H 415.3206 C28H50O5 M+H 467.3731 C27H46O M+H 387.3621 C45H76NO8P M+H 790.5381 C41H71O8P M+H 723.4959 C59H102O6 M+H 907.7749 C42H76NO8P M+H 754.5381 C22H36O2 M+H 333.2788 C53H92O7 M+H 841.6915 C46H80NO7P M+H 790.5745 C47H95N2O6P M+H 815.7000 C46H86NO7P M+H 796.6214 C53H96O6 M+H 829.7279 C26H52NO6P M+H 506.3605 C48H78NO8P M+H 828.5537 C45H69O8P M+H 769.4802 C57H98O6 M+H 879.7436 C45H91N2O6P M+H 787.6687 C47H82NO8P M+H 820.5850 C36H73NO3 M+H 568.5663 C23H35NO2 M+H 358.2740 C50H82NO8P M+H 856.5850 C29H50O2 M+H 431.3883 C39H71O7P M+H 683.5010 C18H33NO M+H 280.2635 C22H36O4 M+H 365.2686 C44H80NO7P M+H 766.5745 C27H49NO2 M+H 420.3836 C55H94O6 M+H 851.7123 C47H82NO8P M+H 820.5850 C21H41NO4 M+H 372.3108 C27H44O3 M+H 417.3363 C43H76NO7P M+H 750.5432 C26H42O M+H 371.3308 C44H86NO8P M+H 788.6163 C44H86NO8P M+H 788.6163 C44H85NO11S M+H 836.5916 C55H96O6 M+H 853.7279 C21H40O4 M+H 357.2999 C42H84NO8P M+H 762.6007 C39H78NO8P M+H 720.5537 C27H53NO4 M+H 456.4047 C21H37NO2 M+H 336.2897 C25H47NO4 M+H 426.3577 C44H84NO6P M+H 754.6109 C47H76NO8P M+H 814.5381 C33H67O7P M+H 607.4697 C44H84NO6P M+H 754.6109 C59H102O6 M+H 907.7749 C46H82NO7P M+H 792.5901 C34H67NO3 M+H 538.5193 C41H74NO8P M+H 740.5224 C47H80NO8P M+H 818.5694 C36H71NO3 M+H 566.5506 C28H48O5 M+H 465.3574

339

C47H73O8P M+H 797.5115 C37H72NO8P M+H 690.5068 C37H68O3 M+H 561.5241 C45H78NO7P M+H 776.5588 C23H36O3 M+H 361.2737 C46H80NO7P M+H 790.5745 C39H68O5 M+H 617.5139 C57H96O6 M+H 877.7279 C47H95N2O6P M+H 815.7000 C47H78NO8P M+H 816.5537 C25H45NO2 M+H 392.3523 C44H84NO7P M+H 770.6058 C53H92O7 M+H 841.6915 C37H68O3 M+H 561.5241 C57H102O6 M+H 883.7749 C22H39NO2 M+H 350.3053 C40H77NO12S M+H 796.5239 C43H69O8P M+H 745.4802 C44H80NO7P M+H 766.5745 C41H76NO8P M+H 742.5381 C27H51NO4 M+H 454.3890 C57H102O6 M+H 883.7749 C25H49NO4 M+H 428.3734 C43H80NO8P M+H 770.5694 C27H46O M+H 387.3621 C41H69O8P M+H 721.4802 C41H80NO8P M+H 746.5694 C59H98O6 M+H 903.7436 C21H37NO2 M+H 336.2897 C50H82NO8P M+H 856.5850 C44H86NO7P M+H 772.6214 C23H36O3 M+H 361.2737 C27H42O3 M+H 415.3206 C35H69O7P M+H 633.4853 C42H76NO8P M+H 754.5381 C46H82NO7P M+H 792.5901 C57H98O6 M+H 879.7436 C39H70O5 M+H 619.5296 C40H76NO8P M+H 730.5381 C42H81NO3 M+H 648.6289 C43H76NO7P M+H 750.5432 C43H82NO8P M+H 772.5850 C26H52NO7P M+H 522.3554 C26H42O M+H 371.3308 C19H38O4 M+H 331.2842 C39H68O5 M+H 617.5139 C45H78NO7P M+H 776.5588 C29H48O2 M+H 429.3727 C42H80NO7P M+H 742.5745 C45H80NO8P M+H 794.5694 C59H100O6 M+H 905.7592 C26H42O2 M+H 387.3257 C43H82NO8P M+H 772.5850 C47H73O8P M+H 797.5115 C35H69O7P M+H 633.4853 C45H76NO6P M+H 758.5483 C47H80NO8P M+H 818.5694 C42H81NO12S M+H 824.5552 C48H78NO8P M+H 828.5537 C27H40O2 M+H 397.3101 C42H81NO3 M+H 648.6289 C43H71O8P M+H 747.4959 C43H69O8P M+H 745.4802 C39H78NO7P M+H 704.5588 C43H73O8P M+H 749.5115 C42H80NO7P M+H 742.5745 C41H69O8P M+H 721.4802 C26H40O2 M+H 385.3101 C44H84NO7P M+H 770.6058 C45H73O8P M+H 773.5115 C25H42O4 M+H 407.3155 C25H47NO5 M+H 442.3527 C41H76NO8P M+H 742.5381 C23H45NO5 M+H 416.3370 C47H82O7 M+H 759.6133 C37H69O7P M+H 657.4853 C28H48O4 M+H 449.3625 C46H86NO8P M+H 812.6163 C53H94O6 M+H 827.7123 C42H82NO9P M+H 776.5800 C41H78NO7P M+H 728.5588 C47H76NO8P M+H 814.5381

340

C43H80NO8P M+H 770.5694 C34H63NO3 M+H 534.4880 C26H46O6 M+H 455.3367 C38H74NO8P M+H 704.5224 C45H76NO6P M+H 758.5483 C22H37NO2 M+H 348.2897 C38H77NO3 M+H 596.5976 C37H66O5 M+H 591.4983 C27H40O2 M+H 397.3101 C38H78NO7P M+H 692.5588 C22H39NO2 M+H 350.3053 C23H46NO7P M+H 480.3084 C39H78NO7P M+H 704.5588 C21H22O7 M+H 387.1438 C22H37NO2 M+H 348.2897 C57H104O6 M+H 885.7905 C38H75NO2 M+H 578.5870 C45H82NO8P M+H 796.5850 C47H74O2 M+H 671.5761 C53H94O6 M+H 827.7123 C59H98O6 M+H 903.7436 C19H40O3 M+H 317.3050 C57H96O6 M+H 877.7279 C19H22O7 M+H 363.1438 C28H60NO6P M+H 538.4231 C32H50O7 M+H 547.3629 C23H38O4 M+H 379.2842 C37H71O7P M+H 659.5010 C24H37NO2 M+H 372.2897 C42H86NO7P M+H 748.6214 C46H86NO8P M+H 812.6163 C19H20O7 M+H 361.1281 C33H67O7P M+H 607.4697 C26H52NO7P M+H 522.3554 C34H63NO3 M+H 534.4880 C41H73O7P M+H 709.5166 C50H80NO8P M+H 854.5694 C43H73O8P M+H 749.5115 C38H78NO7P M+H 692.5588 C41H71O7P M+H 707.5010 C37H72NO8P M+H 690.5068 C40H77NO12S M+H 796.5239 C54H82O4 M+H 795.6285 C48H86NO8P M+H 836.6163 C24H50NO8P M+H 512.3346 C41H76NO7P M+H 726.5432 C55H94O6 M+H 851.7123 C23H43NO4 M+H 398.3264 C36H64O4 M+H 561.4877 C24H37NO2 M+H 372.2897 C21H41NO4 M+H 372.3108 C23H41O6P M+H 445.2713 C25H45NO4 M+H 424.3421 C25H45O4P M+H 441.3128 C45H71O8P M+H 771.4959 C20H24O7 M+H 377.1594 C42H81NO12S M+H 824.5552 C46H80NO9P M+H 822.5643 C48H93O12P M+H 893.6477 C53H94O7 M+H 843.7072 C41H68O5 M+H 641.5139 C21H24O7 M+H 389.1594 C41H73O7P M+H 709.5166 C39H81N2O6P M+H 705.5905 C43H71O8P M+H 747.4959 C45H71O8P M+H 771.4959 C39H76NO7P M+H 702.5432 C35H67O7P M+H 631.4697 C39H78NO8P M+H 720.5537 C39H76NO7P M+H 702.5432 C19H35NO2 M+H 310.2740 C18H20O7 M+H 349.1281 C38H68O4 M+H 589.5190 C43H80NO7P M+H 754.5745 C20H24O7 M+H 377.1594 C18H18O7 M+H 347.1125 C44H85NO11S M+H 836.5916 C15H14O7 M+H 307.0812 C45H80NO8P M+H 794.5694 C45H76NO7P M+H 774.5432 C20H39NO2 M+H 326.3053 C26H50NO7P M+H 520.3397

341

C41H70O5 M+H 643.5296 C50H80NO8P M+H 854.5694 C57H94O7 M+H 891.7072 C44H74NO8P M+H 776.5224 C26H56NO6P M+H 510.3918 C15H10O4 M+H 255.0651 C23H46NO7P M+H 480.3084 C54H82O4 M+H 795.6285 C35H67O7P M+H 631.4697 C18H20O6 M+H 333.1332 C29H48O2 M+H 429.3727 C23H41NO2 M+H 364.3210 C45H82NO8P M+H 796.5850 C39H80NO7P M+H 706.5745 C18H18O6 M+H 331.1176 C22H28O7 M+H 405.1907 C26H50NO7P M+H 520.3397 C42H83NO3 M+H 650.6445 C41H76NO7P M+H 726.5432 C16H12O4 M+H 269.0808 C44H81NO7 M+H 736.6085 C44H76NO9P M+H 794.5330 C41H71O7P M+H 707.5010 C39H69O7P M+H 681.4853 C26H54NO6P M+H 508.3761 C20H18O8 M+H 387.1074 C45H73O8P M+H 773.5115 C45H82NO7P M+H 780.5901 C15H10O4 M+H 255.0651 C18H18O8 M+H 363.1074 C45H67O8P M+H 767.4646 C41H68O5 M+H 641.5139 C29H50O2 M+H 431.3883 C28H54NO8P M+H 564.3659 C29H46F2O3 M+H 481.3487 C20H20O7 M+H 373.1281 C23H48NO7P M+H 482.3241 C45H80NO7P M+H 778.5745 C39H67O8P M+H 695.4646 C27H32F6O3 M+H 519.2328 C42H82NO9P M+H 776.5800 C46H76NO8P M+H 802.5381 C49H93O14P M+H 937.6375 C48H97O9P M+H 849.6943 C45H76NO7P M+H 774.5432 C23H39NO M+H 346.3104 C37H69O7P M+H 657.4853 C27H52NO8P M+H 550.3503 C42H86NO7P M+H 748.6214 C59H100O6 M+H 905.7592 C23H38O3 M+H 363.2893 C41H80NO7P M+H 730.5745 C43H87N2O6P M+H 759.6374 C41H82NO7P M+H 732.5901 C27H46O3 M+H 419.3519 C43H87N2O6P M+H 759.6374 C22H30O M+H 311.2369 C26H54NO6P M+H 508.3761 C42H83NO3 M+H 650.6445 C34H69NO3 M+H 540.5350 C51H90O7 M+H 815.6759 C42H81NO11S M+H 808.5603 C27H49NO4 M+H 452.3734 C37H67O7P M+H 655.4697 C39H81N2O6P M+H 705.5905 C29H40O4 M+H 453.2999 C37H71O7P M+H 659.5010 C31H59O12P M+H 655.3817 C19H20O7 M+H 361.1281 C48H86NO7P M+H 820.6214 C19H40O3 M+H 317.3050 C39H74NO7P M+H 700.5275 C37H76NO7P M+H 678.5432 C41H72NO8P M+H 738.5068 C46H78NO10P M+H 836.5436 C17H14O4 M+H 283.0964 C23H41O6P M+H 445.2713 C27H45NO M+H 400.3574 C50H99O10P M+H 891.7048 C25H50NO7P M+H 508.3397 C25H20O7 M+H 433.1281 C29H46F2O3 M+H 481.3487

342

C45H74NO8P M+H 788.5224 C19H20O8 M+H 377.1231 C18H20O7 M+H 349.1281 C17H18O6 M+H 319.1176 C49H82NO8P M+H 844.5850 C27H42O4 M+H 431.3155 C41H80NO7P M+H 730.5745 C53H92O6 M+H 825.6966 C43H80NO7P M+H 754.5745 C29H49NO4 M+H 476.3734 C48H86NO8P M+H 836.6163 C17H18O7 M+H 335.1125 C37H66O5 M+H 591.4983 C19H24O7 M+H 365.1594 C21H44O3 M+H 345.3363 C43H82NO7P M+H 756.5901 C48H84NO7P M+H 818.6058 C19H35NO2 M+H 310.2740 C27H47NO2 M+H 418.3679 C37H76NO7P M+H 678.5432 C16H16O7 M+H 321.0968 C60H94O6 M+H 911.7123 C45H78NO6P M+H 760.5639 C40H74NO8P M+H 728.5224 C17H12O4 M+H 281.0808 C41H70O5 M+H 643.5296 C27H40O3 M+H 413.3050 C39H72O3 M+H 589.5554 C20H37NO2 M+H 324.2897 C25H48NO7P M+H 506.3241 C27H42O4 M+H 431.3155 C39H66O5 M+H 615.4983 C25H47NO5 M+H 442.3527 C25H38O4 M+H 403.2842 C52H80NO8P M+H 878.5694 C40H78NO7P M+H 716.5588 C45H72NO8P M+H 786.5068 C53H90O7 M+H 839.6759 C27H52NO8P M+H 550.3503 C52H80NO8P M+H 878.5694 C23H39NO M+H 346.3104 C44H86NO6P M+H 756.6265 C53H92O6 M+H 825.6966 C27H47NO2 M+H 418.3679 C39H74NO7P M+H 700.5275 C24H48NO7P M+H 494.3241 C53H90O7 M+H 839.6759 C21H39NO4 M+H 370.2951 C41H82NO8P M+H 748.5850 C31H60NO8P M+H 606.4129 C45H80NO7P M+H 778.5745 C46H93N2O6P M+H 801.6844 C36H68O4 M+H 565.5190 C22H30O M+H 311.2369 C26H42O2 M+H 387.3257 C45H72NO8P M+H 786.5068 C17H28O8 M+H 361.1857 C48H84NO7P M+H 818.6058 C27H45NO2 M+H 416.3523 C48H93O12P M+H 893.6477 C23H43NO5 M+H 414.3214 C41H66O3 M+H 607.5084 C26H40O2 M+H 385.3101 C48H77O10P M+H 845.5327 C49H84NO8P M+H 846.6007 C28H36N2O7 M+H 513.2595 C18H20O6 M+H 333.1332 C49H82NO8P M+H 844.5850 C24H48NO7P M+H 494.3241 C43H72NO8P M+H 762.5068 C41H82NO7P M+H 732.5901 C47H82O7 M+H 759.6133 C27H45NO M+H 400.3574 C38H77N2O6P M+H 689.5592 C57H104O6 M+H 885.7905 C23H40NO7P M+H 474.2615 C27H43NO3 M+H 430.3315 C21H35NO M+H 318.2791 C33H58O4 M+H 519.4407 C16H24O8 M+H 345.1544 C39H66O5 M+H 615.4983 C20H37NO2 M+H 324.2897

343

C47H93O8P M+H 817.6680 C20H22O7 M+H 375.1438 C39H80NO7P M+H 706.5745 C43H84NO8P M+H 774.6007 C19H22O7 M+H 363.1438 C18H18O6 M+H 331.1176 C57H94O6 M+H 875.7123 C49H84NO8P M+H 846.6007 C43H82NO7P M+H 756.5901 C20H26O7 M+H 379.1751 C28H52NO7P M+H 546.3554 C29H56NO8P M+H 578.3816 C23H41NO2 M+H 364.3210 C25H43NO4 M+H 422.3264 C53H94O7 M+H 843.7072 C46H78NO10P M+H 836.5436 C42H81NO11S M+H 808.5603 C23H44NO7P M+H 478.2928 C44H81NO8 M+H 752.6035 C45H74NO8P M+H 788.5224 C23H43NO4 M+H 398.3264 C24H39NO2 M+H 374.3053 C28H36N2O7 M+H 513.2595 C16H16O6 M+H 305.1019 C20H22O7 M+H 375.1438 C55H102O6 M+H 859.7749 C48H86NO7P M+H 820.6214 C24H40O4 M+H 393.2999 C34H69NO3 M+H 540.5350 C27H40O3 M+H 413.3050 C20H26O7 M+H 379.1751 C45H82NO7P M+H 780.5901 C57H100O7 M+H 897.7541 C40H74NO8P M+H 728.5224 C40H75NO8 M+H 698.5565 C39H72O3 M+H 589.5554 C44H78NO10P M+H 812.5436 C44H76NO9P M+H 794.5330 C44H86NO6P M+H 756.6265 C19H24O7 M+H 365.1594 C55H96O7 M+H 869.7228 C37H64O5 M+H 589.4826 C50H84NO8P M+H 858.6007 C24H47NO4 M+H 414.3577 C23H34O2 M+H 343.2631 C22H22O11 M+H 463.1234 C15H12O4 M+H 257.0808 C17H18O7 M+H 335.1125 C16H16O6 M+H 305.1019 C34H64O4 M+H 537.4877 C40H78NO7P M+H 716.5588 C41H66O5 M+H 639.4983 C43H72O5 M+H 669.5452 C37H73O7P M+H 661.5166 C43H84NO8P M+H 774.6007 C44H79O9P M+H 783.5534

344

C50H78NO8P M+H 852.5537 C33H65O7P M+H 605.4540 C50H97O12P M+H 921.6790 C16H26O5 M+H 299.1853 C20H32O4 M+H 337.2373 C38H77N2O6P M+H 689.5592 C30H38N2O7 M+H 539.2751 C46H80NO6P M+H 774.5796 C23H44NO7P M+H 478.2928 C53H98O6 M+H 831.7436 C16H12O4 M+H 269.0808 C48H90NO7P M+H 824.6527 C25H38O4 M+H 403.2842 C25H48NO7P M+H 506.3241 C50H86NO8P M+H 860.6163 C47H78NO8P M+H 816.5537 C18H14O4 M+H 295.0964 C38H74NO8P M+H 704.5224 C42H76NO9P M+H 770.5330 C21H22O7 M+H 387.1438 C37H70O5 M+H 595.5296 C31H41NO3 M+H 476.3159 C27H32F6O3 M+H 519.2328 C34H66O2 M+H 507.5135 C55H102O6 M+H 859.7749 C17H14O4 M+H 283.0964 C37H68O5 M+H 593.5139 C28H54NO8P M+H 564.3659 C18H18O7 M+H 347.1125 C39H69O7P M+H 681.4853 C47H84NO8P M+H 822.6007 C43H72NO7P M+H 746.5119 C47H91O12P M+H 879.6321 C21H43O6P M+H 423.2870 C46H81O10P M+H 825.5640 C24H40NO9P M+H 518.2513 C27H47NO M+H 402.3730 C42H78NO7P M+H 740.5588 C59H96O6 M+H 901.7279 C43H72NO8P M+H 762.5068 C26H45NO M+H 388.3574

345

C42H76NO7P M+H 738.5432 C23H45NO5 M+H 416.3370 C44H84NO9P M+H 802.5956 C18H37NO4S M+H 364.2516 C55H92O6 M+H 849.6966 C43H70NO7P M+H 744.4962 C46H76NO7P M+H 786.5432 C37H64N2O6 M+H 633.4837 C31H54O4 M+H 491.4094 C46H93N2O6P M+H 801.6844 C36H71O8P M+H 663.4959 C36H72NO6P M+H 646.5170 C23H35NO2 M+H 358.2740 C36H67NO8 M+H 642.4939 C21H24O7 M+H 389.1594 C41H66O3 M+H 607.5084 C40H78NO9P M+H 748.5487 C46H88NO8P M+H 814.6320 C45H85O10P M+H 817.5953 C37H67O7P M+H 655.4697 C43H66O5 M+H 663.4983 C16H10O5 M+H 283.0601 C30H58NO8P M+H 592.3972 C26H32O4 M+H 409.2373 C17H10O4 M+H 279.0651 C38H76NO9P M+H 722.5330 C21H20O11 M+H 449.1078 C29H49NO4 M+H 476.3734 C27H38O M+H 379.2995 C42H80NO9P M+H 774.5643 C25H50NO7P M+H 508.3397 C15H31O7P M+H 355.1880 C43H70O5 M+H 667.5296 C48H77O10P M+H 845.5327 C39H67O7P M+H 679.4697 C38H75NO3 M+H 594.5819

346