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Metabolomics & Lipidomics: From Discovery to Routine Applications

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Presenter: Giuseppe Astarita, Ph.D., Principal Scientist, Waters Corp, Adjunct Professor, Georgetown University
A number of technological advancements have enhanced our ability to conduct metabolomics and lipidomics experiments. State-of-the-art chromatography, ionization sources, and MS technology combined with powerful informatics solutions provide a comprehensive set of tools to analyze complex mixtures of lipids and polar metabolites in biological samples. In this presentation, I will illustrate current workflows for metabolomics & lipidomics, including untargeted and targeted approaches, for discovery and routine applications.
This presentation was given at the 11th International Conference of the Metabolomics Society (Metabolomics 2015, #metsoc2015 on Twitter), June 29, 2015, in San Francisco.

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Metabolomics & Lipidomics: From Discovery to Routine Applications

  1. 1. ©2015 Waters Corporation 1 Metabolomics and Lipidomics: from Discovery to Routine Applications 11th International Conference of the Metabolomics Society June 29th 2015 to July 2nd 2015 Giuseppe Astarita Principal Scientist, Health Sciences Waters Corp, USA
  2. 2. ©2015 Waters Corporation 2  Metabolomics and Lipidomics Application Notebook – Waters Corporation, April 2015. Literature part number 720005245EN – http://www.waters.com/waters/library.htm?cid=511436&lid=134841740 Compilation of Recent Work
  3. 3. In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) O O HO R1 O O N + P OH O O R2 Phosphotadylcholine (PC) sn-1 sn-2 sn-1-H20 sn-2-H20 Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) 1 2 3 4 Metabolomics Approaches
  4. 4. In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) O O HO R1 O O N + P OH O O R2 Phosphotadylcholine (PC) sn-1 sn-2 sn-1-H20 sn-2-H20 Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) 1 2 3 4 Metabolomics Approaches
  5. 5. Measure (Mass Spectrometry) Separate (Chromatography; Ion Mobility) Process and Mine (Informatics and Statistics) Untargeted Metabolomics and Lipidomics
  6. 6. Metabolomics: UPLC Separation Paglia G.et al. Anal Chem 2014
  7. 7. Low Energy High Energy Metabolomics: APGC Separation Arabidopsis Courtesy of Prof Vladimir Shulaev
  8. 8. Lipidomics: UPLC Separation ChoE & TGPC, SM, PG, PE lysophospolipids SM, DG, ChoE PC, PG,PI, PS, PE Free Fatty Acids Damen C., et al. Journal Lipid Research 2014
  9. 9. Lipidomics: HILIC/SFC Mouse heart extract Miroslav Lísa and Michal Holčapek Anal Chem. 2015 Jun 20
  10. 10. In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) O O HO R1 O O N + P OH O O R2 Phosphotadylcholine (PC) sn-1 sn-2 sn-1-H20 sn-2-H20 Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) 1 2 3 4 Metabolomics Approaches
  11. 11. Drift time m/z Ceramide species Drift time (ms) Drift time (ms) p _ _ _ _ _ Drifttime (ms) Retention time (min) RelativeIntensity (%) 0 2 4 6 8 10 12 14 16 18 20 ChoE & TG PC, SM, PG, PElysophospholipids Orthogonal Coordinates Paglia G., et al. Anal Bioanal Chem 2015
  12. 12. Paglia G.et al. Anal Chem 2015; Paglia et al. Anal Chem 2014 Ion Mobility Separation: Hybrid Q-TOF Poster 353 – The Analysis of Bile Acids: Enhancement of Specificity Using Travelling Wave IMS-QTof Mass Spectrometry
  13. 13. ©2015 Waters Corporation 14 DIA with IMS (HDMSE) Paglia G., et al. Anal Bioanal Chem 2015
  14. 14. Composite ion map after peak picking Individual ion maps Data alignment and peak picking ANOVA filtering and Multivariate Statistics Database search Filtering by ANOVA P value Control Radiation Control Radiation Tentative Identifications 1. 2. 3. 4. 5. Data Processing and Mining with Progenesis QI Software Laiakis E. et al, J Proteome Res. 2014 Sep
  15. 15. 0 20 40 60 80 100 120 140 160 180 5 6 7 8 9 10 11 12 13 14 15 identifications ppm m/z error HMDB In house database no RT In house database with RT <0.6 min Database Search Using Orthogonal Coordinates
  16. 16. Database Search Using Orthogonal Coordinates HDMSE <25 fragments HDMSE High Energy HDMSE Low Energy 750.5422 750.5422 303.2324 (FA 20:4) O HO O O NH2 P HO O O 303.2324 (FA 20:4) % % m/z200 1400 With Ion mobility separation Co-eluting ions Fragments Transferred to TOF-MS Fragmentation
  17. 17. Methodological References: Untargeted A Facile Database Search Engine for Metabolite Identification and Biomarker Discovery in Metabolomics Development of a Metabolomic Assay for the Analysis of Polar Metabolites Using HILIC UPLC/QTof MS Lipid Separation Using UPLC with Charged Surface Hybrid Technology (pending publication)
  18. 18. In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) O O HO R1 O O N + P OH O O R2 Phosphotadylcholine (PC) sn-1 sn-2 sn-1-H20 sn-2-H20 Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) 1 2 3 4 Metabolomics Approaches
  19. 19. Analyze (Software) Measure (Tandem Mass Spectrometry) Separate (Chromatography) Sample Preparation (extraction Liq/Liq; SPE) Targeted Metabolomics
  20. 20. Targeted Metabolic Profiling: Metabolomics kits Metabolite group No. of metabolites Amino acids and Biogenic amines 40 Acylcarnitines 40 Lyso-phosphatidylcholines 14 Phosphatidylcholines 74 Sphingomyelins 14 Hexose 1 Total 183 + Laiakis E. et al, J Proteome Res. 2014 Sep  Biocrates AbsoluteIDQ p180 Kit  Waters Xevo TQ-S Mass Spectrometer
  21. 21. Eicosanoids: Bioactive Oxygenated PUFAs Omega-6 metabolites: pro-inflammatory Omega-3 metabolites: anti-inflammatory Astarita et al. Biochim Biophys Acta. 2015 Apr;1851(4):456-468
  22. 22. Plasma sample Add internal standard mix Load Inject into UPLC/MS-MS SPE clean up Multiplexed Assay for Eicosanoids Profiling Selected Lipid Internal Standard
  23. 23. Methodological References: Targeted  Targeted Lipidomics of Oxylipins (Oxygenated Fatty Acids)  Targeted Lipidomics Using the ionKey/MS System  Targeted Metabolomics Using the UPLC/MS- based AbsoluteIDQ p180 Kit
  24. 24. In situ; Imaging Scanning along x and y axes using high-res instruments (spatial information; molecular histology) Targeted Monitoring selected ions in high-res or nominal mass (quantitative; sensitive; requires internal standards) Structural Elucidation High-res instruments with fragmentation capabilities (chemical structure) O O HO R1 O O N + P OH O O R2 Phosphotadylcholine (PC) sn-1 sn-2 sn-1-H20 sn-2-H20 Untargeted Scanning for differences using high-res instruments (semi-quantitative; statistics-based; hypothesis-generating) 1 2 3 4 Metabolomics Approaches
  25. 25. ©2015 Waters Corporation 26
  26. 26. ©2015 Waters Corporation 27 100 700 1500 m/z 50 150 DriftTime(bins) + + + Ion mobility MS Brain slice Excitation source (i.e., laser, ESI) + Scanning tissues along the axes desorption/ionization MOLECULAR HISTOLOGY Paglia G.et al. Anal Chem 2015 MS Imaging with Ion Mobility
  27. 27. ©2015 Waters Corporation 28 In Situ Metabolomics  REIMS Research System with iKnife for Direct Sampling
  28. 28. Methodological References: MS Imaging  Improved MALDI Imaging Quality and Speed Using the MALDI SYNAPT G2-Si HDMS  Biomarker Discovery Directly from Tissue Xenograph Using High Definition Imaging MALDI Combined with Multivariate Analysis  Data Independent MALDI Imaging HDMSE for Visualization and Identification of Lipids Directly from a Single Tissue Section  Utility of Desorption Electrospray Ionization (DESI) for Mass Spectrometry Imaging
  29. 29. ©2015 Waters Corporation 30 Peer-Reviewed References
  30. 30. Conclusions Spatial Localization/Real Time Quant/Qual applications Screening and IdentifyO O HO R1 O O N + P OH O O R2 Phosphotadylcholine (PC) sn-1 sn-2 sn-1-H20 sn-2-H20 Chemical Diversity1 2 3 4
  31. 31. ©2015 Waters Corporation 32 Acknowledgements Jon Williams Scott Geromanos Donald Mason Steven Lai Giorgis Isaac Hernando Olivos Emmanuelle Claude Jim Langridge Arthur Moseley Will Thompson David Grant Lochana Menikarachchi Giuseppe Paglia Bernhard O. Palsson Skarphédinn Halldórsson Ottar Rolfsson Peggi Angel Callee Walsh Greg Kilby Thomas Hankemeier Rob Vreeker Katrin Strassburg Albert Joseph Fornace Evagelia C. Laiakis Ralf Bogumil Cornelia Roehring Therese Koal Jing Kang Jennifer McKenzie

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