New Frontiers for MS in Metabolipidomics

New Frontiers for MS in Metabolipidomics Giuseppe Astarita, PhD Principal Scientist Discovery & Life Sciences Milford, MA USA 2011 Waters Corporation 1

Why Metabolipidomics? Biomedical Sciences o Biomarker discovery o Drug Discovery and Development o Microbiology o Personalized medicine Environmental Sciences o Strain fingerprinting and ID o Genetic modifications to improve phenotypes o Pesticide Residue Food Sciences o Nutrients composition o Purity Metabolic engineering o Improvement of metabolic pathways for the production of fuels and chemicals Natural Products o Traditional medicines 2011 Waters Corporation 2

Metabolipidomics DNA Genomics RNA Transcriptomics Proteins Proteomics Environment: diet, pollution, microbiome, drugs Sugars, aa, nucleic acids, toxins Metabolites Lipids Metabolomics Lipidomics Metabolites offer an instantaneous snapshot of the physiology of the cell, reflecting both genetic information and environmental influences. 2011 Waters Corporation 3

Metabolipidomics in the context of systems biology What can happen Genomics 2 x 10 4 genes What appears to be happening Transcriptomics 10 5 transcripts What makes it happen Proteomics 10 6 proteins What has happened And is happening Metabolipidomics 10 4 molecular species Phenotype 2011 Waters Corporation 4

Chemical diversity 2011 Waters Corporation 5

Interconnected networks R' H P H N P R R' H H NH 2 PC NAT PE H R' H H H H P H P H H N H N Lyso-NAPE PLA 1, ABHD-4 GP-anandamide PLA 2, ABHD-4 Lyso-PLD P R R' H H PDE LPA H PA H N H N NAPE NAPE-PLD Anandamide FAAH P PLC H - P H N LX, CYP450, CX-2 PAEA H Arachidonic acid xygenated derivatives 2011 Waters Corporation 6

Complexity of metabolic networks 2011 Waters Corporation 7

Metabolipidomics Hardware 2011 Waters Corporation 8

How to screen and characterize complex mixtures of metabolites? 2011 Waters Corporation 9

UPLC separation UPLC HPLC HPLC UPLC HPLC UPLC 2011 Waters Corporation 10

MS for metabolipidomics Xevo G2 QTof Xevo TQ-S SYNAPT G2-S 2011 Waters Corporation 11

High resolution mass 2011 Waters Corporation 12

Ion Mobility separation Q IN MBILITY CELL TF CLLISIN CELLS 2011 Waters Corporation 13

Ion Mobility separation IM refers to the motion of gas-phase ions in the presence of gas (nitrogen, for instance) within a pressurized chamber (between 1 and 760 Torr), which is governed principally by the collision frequency between the ion and the gas 2011 Waters Corporation 14

Time Aligned Parallel Fragmentation (TAP) Single ion Intensity Intensity Intensity m/z m/z m/z Drift time Drift time Precursor ion are isolated in the quadrupole Precursor ion are fragmented into product ions in the trap cell Product ions are separated in the drift tube by ion mobility Product ions are fragmented in the transfer cell 2011 Waters Corporation 15

MS E : Quant/Qual Data Acquisition 2011 Waters Corporation 16

MS E Spectral Information PC (18:2/16:0) PC 16:0/18:2 2011 Waters Corporation 17

Metabolipidomics Software 2011 Waters Corporation 18

Comparative metabolipidomics You look different! I suspect that my metabolites are different from yours! 2011 Waters Corporation 19

Metabolipidomics strategies 2011 Waters Corporation 20

Metabolipidomics strategies A)Untargeted Hypothesis generating or discovery-driven/exploratory ( No initial hypothesis ; unbiased; -omics based) Global profiling screen as many lipids as possible B) Targeted Hypothesis driven monitoring selected ions 2011 Waters Corporation 21

Metabolipidomics workflow CNTRL Tissue or cells Sample prep Lipid extraction MS analysis Data processing and mining Identification & quantification DISEASE Tissue or cells Sample prep Lipid extraction MS analysis Data processing and mining Identification & quantification 2011 Waters Corporation 22

Research Design Untargeted analysis Generate hypothesis Test the hypothesis Targeted analysis 2011 Waters Corporation 23

Software for untargeted: MarkerLynx 2011 Waters Corporation 24

Software for untargeted: MarkerLynx MarkerLynx allows even a novice user to perform analysis easily without the need to become an expert in statistics. 2011 Waters Corporation 25

Statistical analysis and visualization 2 3 4 1 Spectrum (observation) becomes a point in PCA Scores plot 3_185.049 Variables (m/z_rt) shown in PCA Loadings Plot 3_213.043 These plots allow trends in the sample spectra to be interpreted in terms of m/z 26 2011 Waters Corporation 26

Statistical analysis and visualization 2011 Waters Corporation 27

Structural Elucidation: MassFragment 2011 Waters Corporation 28

Software for targeted: TargetLynx Peak selected into TargetLynx method editor 2011 Waters Corporation 29

Software for targeted: TargetLynx Sample peak Internal Standard peak 2011 Waters Corporation 30

Software for targeted: TargetLynx Targeteting > 215 lipid species Lipid Class Polarity MRM Time Window(min) Cone Voltage (V) Collision Energy (V) Hexosylceramide (MonoHexCer) + 0-2 20 30 Phosphatidylglycerol (PG) - 1-3 55 45 DiHexosylceramide (DiHexCer) + 2-4 20 30 Phosphatidylinositol (PI) - 3-5 48 30 Phosphatidylethanolamine (PE) - 4-6 48 40 Phosphatidylcholine (PC) + 5-7 36 30 Lyso-Phosphatidylinositol (LPI) - 5-7 48 30 Lyso-Phosphatidylethanolamine (LPE) - 6-8 36 24 Sphingomyelin (SM) + 7-9 36 24 Lyso-Phosphatidylcholine (LPC) + 8-10 42 26 2011 Waters Corporation 31

Demo time! 2011 Waters Corporation 32

Metabolipidomics Solutions 2011 Waters Corporation 33

Metabolipidomics Solutions 1) Sample preparation 2) nline-separation MS 3) Direct infusion MS 4) Surface-based MS + Ion Mobility 2011 Waters Corporation 34

Metabolipidomics Solutions 1) Sample preparation 2) nline-separation MS 3) Direct infusion MS 4) Surface-based MS + Ion Mobility 2011 Waters Corporation 35

Sample preparation Used in Bioanalysis to remove phospholipids from serum and plasma samples Lipid classes can be selectively eluted (purified) afterwards Better sample preparation = more small molecules detected ASMS Poster 2011 (MP257) 2011 Waters Corporation 36

Metabolipidomics Solutions 1) Sample preparation 2) nline-separation MS 3) Direct infusion MS 4) Surface-based MS + Ion Mobility 2011 Waters Corporation 37

nline-separation MS: LC/MS Metabolipidomics challenges: Thousands of metabolites Many isobaric or isomeric species Many very low-abundance species (pmolar and lower) 2011 Waters Corporation 38

nline-separation MS: LC/MS Metabolipidomics challenges: Thousands of metabolites Many metabolites are isobaric or isomeric species Many metabolites are very low-abundance (pmolar and lower) Solutions: LC can help to purify complex mixture of metabolites increasing sensitivity LC provides a new level of description (retention time, index, factor) 2011 Waters Corporation 39

nline-separation MS: LC/MS 1) UPLC by reversed phase (RP) or hydrophilic interaction chromatography (HILIC) 2) Parallel column regeneration solutions 3) Supercritical fluid chromatography Additional R&D effort: 1) Microfluidics (Triziac) 2) Multi-dimensional LC (e.g., HILIC+RP) 2011 Waters Corporation 40

nline-separation MS: LC/MS 1) UPLC by reversed phase (RP) or hydrophilic interaction chromatography (HILIC) 2) Parallel column regeneration solutions 3) Supercritical fluid chromatography Additional R&D effort: 1) Microfluidics (Triziac) 2) Multi-dimensional LC (e.g., HILIC+RP) 2011 Waters Corporation 41

Reversed phase vs HILIC Reversed phase - Separation by acyl chain and numbers of double bonds PC, SM, PG, PE, ChoE lyso-pc, lyso-pe. J. Proteome Res., 2010, 9 (5), pp 2377 2389 DG & TG HILIC - Separation by polar groups C17 Cer 0.59 DMPG 2.27 DMPE 4.63 DMPC 6.08 L-SM 7.78 20:0 Lyso PC 8.20 2011 Waters Corporation 42

Reversed phase/hdms E PI, PC, PG, PE TG CE lysophospolipids SM, DG 2011 Waters Corporation 43

HDMS chromatogram: Drift Time vs Retention Time Retention time (min) 2011 Waters Corporation 44

Isomers separation: Gangliosides GD 1a GD 1b 2011 Waters Corporation 45

HILIC/HDMS E Cer d18:1/16:0 Cer d18:1/18:0 Cers Cer d18:1/24:1 Drift time PC 18:0/18:1 PC 16:0/18:1 PC 14:0/18:1 PCs Drift time SMs BEH HILIC 1.7 µm, 2.1x100 mm Retention time (min) 2011 Waters Corporation 46

Molecular landscapes Ion mobility separation m/z Ceramide species Drift time 2011 Waters Corporation 47

MS E for chemical characterization Low collision energy = precursor ions High collision energy = product ions 2011 Waters Corporation 48

Mapping brain metabolipidome Brain metabolipidome Drift time Ceramides PCs SMs Ion mobility separation Drift time Retention time (min) 2011 Waters Corporation 49

nline-separation MS: LC/MS 1) LC by reversed phase (RP) or hydrophilic interaction chromatography (HILIC) 2) Parallel column regeneration solutions 3) Supercritical fluid chromatography 2011 Waters Corporation 50

Parallel Column Regeneration Q. How can we reduce analysis time by half? A. Parallel column regeneration. We can set up one column to perform an analytical separation, while the second column is re-equilibrated preparing for the next injection 2011 Waters Corporation 51

Parallel Column Regeneration 2011 Waters Corporation 52

Parallel Column Regeneration 2011 Waters Corporation 53

Parallel Column Regeneration Typical UPLC UPLC with 2D Technology Injection to Injection Cycle Time 12 Minutes 8 Minutes Samples Analyzed in 24 Hours 120 180 8 Minute Separation and 4 Minute Re-Equilibration 50% Increase in Throughput 2011 Waters Corporation 54

nline-separation MS: LC/MS 1) LC by reversed phase (RP) or hydrophilic interaction chromatography (HILIC) 2) Parallel column regeneration solutions 3) Supercritical fluid chromatography 2011 Waters Corporation 55

Supercritical fluid chromatography:upc2 Supercritical fluids are highly compressed gases which combine properties of gases and liquids (exhibit low viscosity and high solute diffusivity) Advantages: lower backpressure shorter reequilibration times higher throughput reducing the use of toxic solvents and the cost of analyses 2011 Waters Corporation 56

Supercritical fluid chromatography: UPC2 8x10 7 PC 6x10 7 Intensity 4x10 7 2x10 7 PE SM LPC CER PE LPE 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Minutes 2011 Waters Corporation 57

Metabolipidomics Solutions 1) Sample preparation 2) nline-separation MS 3) Direct infusion MS 4) Surface-based MS + Ion Mobility 2011 Waters Corporation 58

Direct infusion - Shotgun metabolipidomics 1) Top down (TF) 2) Bottom up (TQ) Advantages: high throughput Disadvantages: ion-suppression, isomers, isotopes 2011 Waters Corporation 59

Shotgun metabolipidomics Product ion scan Quadrupole 1 Quadrupole 2: collision cell Select precursor ion A B C CID Quadrupole 3 Scan product ions A B C m/z m/z Precursor ion scan Scan precursor X ions B A B C C CID Select product ion C Neutral loss m/z Select precursor X ions B A B C E C CID Scan product ions with an offset mass (e.g., B) X m/z C A C m/z m/z Multiple Reaction Monitoring Select precursor ion A B C CID Select product ion B m/z 2011 Waters Corporation 60 m/z

Shotgun metabolipidomics m/z 184.1 2011 Waters Corporation 61

Metabolipidomics Solutions 1) Sample preparation 2) nline-separation MS 3) Direct infusion MS 4) Surface-based MS + Ion Mobility 2011 Waters Corporation 62

Surface-based MS - Desorption ionization to mass analyzer Excitatory beam Sample Advantages: spatial resolution, no sample prep, fingerprinting Disadvantages: ion suppression, isomers, quantification 2011 Waters Corporation 63

Surface-based MS - Desorption ionization 1) In situ, real-time, rapid screening (e.g., skin, dried blood spots, biopsies, bacteria) 2) Planar chromatography or processed sample (e.g., TLC/PC) 3) MS imaging (e.g., tissue slices) Excitatory beam to mass analyzer Sample 2011 Waters Corporation 64

Direct Analysis in Real Time/HDMS Human sebum 2011 Waters Corporation 65

Direct Analysis in Real Time/HDMS: ion mobility separation of fatty acids C18:0, stearic acid C18:1, oleic acid C18:2, linoleic acid C16:0, palmitic acid C16:1, palmitoleic acid 2011 Waters Corporation 66

Surface-based MS - Desorption ionization 1) In situ, real-time, rapid screening (e.g., skin, dried blood spots, biopsies, bacteria) 2) Planar chromatography or processed sample (e.g., TLC/PC) 3) MS imaging (e.g., tissue slices) DI-MS can be used for scanning through tissues and cells along the x -and y- axes to create topographic maps of the metabolite composition. 2011 Waters Corporation 67

Brain MALDI/MS imaging 20-100 µm 2011 Waters Corporation 68

MALDI/MS imaging 788.6 866.6 734.5 793.4 2011 Waters Corporation 69

MALDI/MS imaging Control diet High-Fat diet 2011 Waters Corporation 70

Whole body MALDI/MS imaging No IMS Longer Drift time Shorter Drift time 2011 Waters Corporation 71

Waters metabolipidomics Thank you for your attention! Visit us at the ASMS 2012!! 2011 Waters Corporation 72

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Time Aligned Parallel Fragmentation (TAP) A C4 C6 C7 C8 PC 16:0/18:0 C5 B C4 C5 C6 C7 C8 C9 C10 C11 PC 16:0/18:1(9Z ) C12 C13 C C6 C7 C9 PC16:0/18:2(9Z,12Z) C5 C8 C10 C11 C12 C13 C14 C4 2011 Waters Corporation 74

Automated lipid identification 2011 Waters Corporation 75

Targeted versus untargeted Phospholipids H P - X PLA 1 AT Lyso-PL H H P - X PIP 2 TAG R 1 H H P - PIP 2 -PLC H P - 3 H H P 3 - TAGL R 2 AT DAG CDP-DAG PL-PLC P H H AT DGL MAGK H H H DAGK P PLD PA AT Lyso-PA PLA 1 H H Lyso-PLD H P - H P - H Lyso-PLC 2-AG MGL, ABDH6, ABDH12 LX, CYP450, CX-2 xygenated derivatives H Arachidonic acid 2011 Waters Corporation 76