1. Identification and Structure Determination of Higher Order Glycosphingolipids via LC-MS/MS M. Cameron Sullards, Ph. D. Georgia Institute of Technology: School of Chemistry and Biochemistry and School of Biology, Atlanta, GA 30332-0363

2. Gene mRNA Protein Post-translational modification(s) Metabolites Cellular functions Membrane lipids and regulation of cell structure/function Membrane (lipid) functions: Energy Structure Transport Signaling Implications for disease: Energy excess /deficit Aberrant cell structure/function Transport defects Incorrect signaling Developmental abnormalities Agonists & other extracellular signals, including nutrients & xenobiotics

3. LIPID MAPS

4. Lipid Metabolites And Pathways Strategy GENOMICS PROTEOMICS METABOLOMICS “LIPIDOMICS”

5. Lipid Metabolites and Pathways Strategy LIPID MAPS GOALS (1)To separate and detect all of the lipids in a specific cell and to discover and characterize any novel lipids that may be present. (2)To quantitate each of the lipid metabolites present and to quantitate the changes in their levels and location during cellular function. (3) To define the biochemical pathways for each lipid and develop lipid maps which define the interaction networks.

6. LIPID MAPS CORES Bioinformatics Neutral Lipids Other Lipids/Structural Lipidomics Sterols Fatty Acids/Eicosanoids Glycerophospholipids Sphingolipids/Gangliosides Lipid Synthesis/Characterization Michael VanNieuwenhze - UCSD Walter Shaw - Avanti Polar Lipids Steven White - UC Irvine Shankar Subramaniam - UCSD Robert C. Murphy - Colorado Christian Raetz - Duke David W. Russell - UTSW Alfred H. Merrill - Georgia Tech H. Alex Brown - Vanderbilt Macrophage Biology Christopher Glass - UCSD Edward A. Dennis - UCSD LC/Mass Spec Robert C. Murphy - Colorado PI Edward A. Dennis - UCSD LIPID MAPS

7. Sphingolipids are the most structurally complex and diverse lipids of eukaryotes HO O O OH OH O O O OH O OH H O HO O OH HO OH H 1 O OH HO OH O HNAc OH HO HO H H Cer Lactosylceramide (LacCer) 3 4 3 1 2 1 1 4 1' N-Acetyl- galactosamine Galactose Glucose AcNH HO 2 C N-Acetyl- Neuraminic acid G M1 G M2 G M3 Glucosylceramide (GlcCer) OH NH D-erythro-sphingosine Ceramide (N-acylsphingosine) O (CH 3 ) 3 NCH 2 CH 2 O-P(O 2 H)-O-Cer Sphingomyelin J. L. W. Thudichum 1884

8. SphinGOMAP (Download available at www.sphingomap.org) SphinGOMAP © (Download available at www.sphingomap.org)www.sphingomap.org

9. MS/MS Methodology  Identify structure specific dissociations unique to various classes SL’s (ie. Cer, GlcCer, LacCer, Gb3, and Gb4)  Optimize ionization and dissociation conditions for all SL’s  Utilize precursor ion & neutral loss scans to identify individual headgroup, base, and fatty acid combinations of endogenous SL’s  Quantify SL’s using internal standards and LC-MS/MS

10. Globoside Gb3 Globoside Gb4

11. (M 1 + H) + * * * Gb3 (M 2 + H) + (M 3 + H) + * = (M + Na) +

12. 4000 Q TRAP TM System – Ion Path Q0Q1 Q2Q3 LIT Curtain Plate Orifice Skimmer LINAC Exit Lens

13. 340u = C22:0 Glc Gal Quad Fragments Quad Scan d18:1 (M 1 + H) +

15. Quad Fragments LIT Scan d18:1 340u = C22:0 Glc Gal (M 1 + H) +

16. Quad Fragments LIT Scan w/ Q 0 Trapping d18:1 340u = C22:0 Glc Gal (M 1 + H) +

17. 4000 Q TRAP TM System – Ion Path Q0Q1 Q2Q3 LIT Curtain Plate Orifice Skimmer LINAC Exit Lens

18. MS/MS/MS 1108.7604.6

19. d18:1 368u = C24:0 Gal Glc (M 2 + H) +

20. d18:1 384u = h24:0 Glc Gal (M 3 + H) +

22. 1024.9 1053.0 1081.1 1125.1 1135.1 C16:0 h24:1 h24:0 C18:0 C20:0 C22:0 h22:0 C24:1 d18:1

23. Gb4 (M 1 + H) + * * * * = (M + Na) +

25. (M 1 + H) + GalNAc Gal Glc 340u = C22:0 d18:1

26. (M 2 + H) + GalNAc Gal Glc368u = C24:0 d18:1

27. (M 3 + H) + GalNAc Gal Glc d18:1 384u = h24:0

28. 1228.0 1256.1 1284.1 C16:0 C18:0 C20:0 C22:0 C24:0 h24:0 h24:1 d18:1

29. LC ESI MS/MS Sample In (Autosampler) Inlet System Ion Source Mass Analyzer (Q1, Q2, Q3) Detector Data System Data out HPLC Relative Ion Abundance 0246810 Time (min) Cer SM LacCer GlcCer

30. Gb3 d18:1 C16:0 C18:0 C20:0 h24:1 h22:0 C24:1 C22:0 h24:0 C24:0

31. Gb4 C20:0 C18:0 C16:0 h24:1 C24:1 C22:0 h22:0 h24:0 C24:0 d18:1

32. Conclusions  Enhanced product ion scans in conjunction w/ Q 0 trapping yield more highly abundant fragment ions enabling detailed structural analysis  Precursor ion scans reveal critical information regarding low abundance globosides with high sensitivity in crude lipid extracts  MS/MS/MS may be used for determination of specific headgroup, long chain base, and fatty acid combinations in globosides  LC in conjunction w/ MS/MS is a powerful tool for separation and identification of complex mixtures of globosides

33. Acknowledgements Prof. Alfred H. Merrill, Jr. Meeyoung Park Anu Koppikar http://www.sphingomap.orgMatreya NIH/NIGMS/Lipid MAPS http://www.lipidmaps.org