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Dual Parallel Mass Spectrometers with 31 P NMR for Sphingolipid Analysis

Dual Parallel Mass Spectrometers with 31 P NMR for Sphingolipid Analysis. Wm. Craig Byrdwell a Richard H. Perry b a Food Composition Laboratory, USDA, ARS, Beltsville Human Nutrition Research Center b Purdue University Dept. of Chemistry. Overview. What are Sphingolipids?

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Dual Parallel Mass Spectrometers with 31 P NMR for Sphingolipid Analysis

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  1. Dual Parallel Mass Spectrometers with 31P NMR for Sphingolipid Analysis Wm. Craig Byrdwella Richard H. Perryb aFood Composition Laboratory, USDA, ARS, Beltsville Human Nutrition Research Center bPurdue University Dept. of Chemistry

  2. Overview • What are Sphingolipids? • Why study Sphingolipids? • What information can APCI-MS provide? • What information can ESI-MS provide? • Why Dual Parallel Mass Spectrometers? • Why use 31P-NMR? What information? • What about quantification?

  3. What are Sphingolipids? They are a FAMILY of molecules based on the ceramide backbone , formed as follows : Sphinganine 3-Ketosphinganine O

  4. What are Sphingolipids? Sphinganine Sphingomyelin ! Ceramide Dihydroceramide

  5. The Sphingolipids we’re interested in: Sphingomyelin (d18:1/18:1 SM) Dihydrosphingomyelin (d18:0/18:1 DSM)

  6. Why Study Sphingomyelin? • Sphingomyelins are major components in the plasma membrane bilayers of cells. • Sphingomyelins have special properties: • They self-associate into ‘membrane rafts’ • They have a higher gel-to-liquid crystalline phase transition temperature than glycerophospholipids • They play crucial roles in cellular signaling: • Sphingomyelinase acts on sphingomyelin to produce ceramide, a potent cell signaling molecule • They have been mischaracterized.

  7. Why Study Sphingomyelin? Results in the past have been mixed… With some agreement, But Substantial disagreement

  8. APCI-MS, MS/MS and MS3 Of Bovine Brain Sphingolipids

  9. 24:1 DSM 22:0 DSM 24:0 DSM 20:0 DSM * See masses at www.Sphingomyelin.com

  10. 24:1 SM 24:0 SM 20:0 SM 22:0 SM * 18:0 DSM See masses at www.Sphingomyelin.com

  11. 18:0 SM [(Cer+H)-2H2O]+ 20:0 SM 16:0 DSM See masses at www.Sphingomyelin.com

  12. SL1 24:1 DSM [Cer-H2O+H]+ [Cer-2H2O+H]+ APCI-MS, MS/MS and MS3 of Bovine Brain Dihydrosphingomyelin [24:1 FA(L)]+ [LCB-H2O]+ [LCB]+ [24:1 FA(S)]+ See masses at www.Sphingomyelin.com

  13. [Cer-2H2O+H]+ [Cer-3H2O+H]+ D14 = CH2 [LCB-H2O]+ [24:1 FA(L)]+ * See masses at www.Sphingomyelin.com

  14. SL2 24:0 SM [Cer-H2O+H]+ [Cer-2H2O+H]+ APCI-MS, MS/MS and MS3 of Bovine Brain Sphingomyelin [24:0 FA(L)]+ [LCB-H2O]+ [24:0 FA(S)]+ [LCB]+ See masses at www.Sphingomyelin.com

  15. [Cer-2H2O+H]+ [24:0 FA(L)]+ [LCB-H2O]+ See masses at www.Sphingomyelin.com

  16. ESI-MS and APCI-MS Simultaneously (Dual Parallel MS) Of Bovine Brain Sphingolipids

  17. APCI-MS ESI-MS 24:1 DSM DPPC 24:1 DSM 22:0 DSM 24:0 DSM 24:0 DSM 22:0 DSM -H2O 20:0 DSM 20:0 DSM [PP]+ +Na -CH2 +Na -H2O

  18. APCI-MS ESI-MS 24:1 SM -H2O 24:1 SM 20:0 SM 24:0 SM 22:0 SM -H2O 24:0 SM 18:0 DSM 18:0 DSM 22:0 SM 20:0 SM 16:0 DSM -H2O

  19. APCI-MS ESI-MS 18:0 SM 18:0 SM -H2O -H2O 20:0 SM 20:0 SM 16:0 SM 16:0 SM +Na -H2O

  20. APCI-MS ESI-MS 24:0 DSM m/z 634.6 24:0 DSM m/z 817.7 24:1 DSM 24:0 SM m/z 632.6 24:1 DSM 24:0 SM m/z 815.7 20:0 DSM m/z 578.5 20:0 DSM m/z 761.6 20:0 SM m/z 576.5 20:0 SM m/z 759.6 18:0 DSM m/z 522.5 18:0 DSM m/z 733.6 18:0 SM m/z 548.5 18:0 SM m/z 731.6

  21. 31P Nuclear Magnetic Resonance (NMR) Spectroscopy Of Sphingolipids

  22. 31P NMR Spectroscopy SM -0.081 DPPC (I.S.) -0.840 DSM +0.133

  23. SM -0.081 DPPC (I.S.) -0.840 DSM +0.133 31P NMR Spectroscopy 1.0143 6.9094 3.0463

  24. 31P NMR Spectroscopy of Bovine Milk SM -0.084 DPPC (I.S.) -0.840 DSM +0.129

  25. SM -0.084 DPPC (I.S.) -0.840 DSM +0.129 31P NMR Spectroscopy of Bovine Milk 0.9853 5.1273 2.3353

  26. Dual Parallel Mass Spectrometry (APCI-MS & ESI-MS) of Bovine Milk Sphingolipids

  27. APCI-MS ESI-MS 24:0 DSM 24:0 DSM m/z 634.6 m/z 817.7 24:1 DSM 24:0 SM m/z 632.6 24:1 DSM 24:0 SM m/z 815.7 22:0 DSM m/z 606.6 22:0 DSM m/z 789.7 22:0 SM m/z 604.6 22:0 SM m/z 787.7 16:0 DSM m/z 522.5 16:0 DSM m/z 705.6 16:0 SM m/z 520.5 16:0 SM m/z 703.6

  28. Quantification of Sphingolipids By APCI-MS and ESI-MS (if time allows)

  29. Bovine Brain Sphingolipid Molecular Species by APCI-MS

  30. Bovine Brain Sphingolipid Molecular Species by APCI-MS

  31. Bovine Brain Sphingolipid Molecular Species by ESI-MS

  32. Bovine Brain Sphingolipid Molecular Species by ESI-MS

  33. Conclusion • Dihydrosphingomyelin is a dietary component! • APCI-MS can provide fragments to identify the backbone and fatty amide. • ESI-MS provides the molecular weight. • Dual Parallel Mass Spectrometers reduce or eliminate ambiguity. • 31P-NMR proves DSM is present.

  34. Thank You for your Attention! Wm. Craig Byrdwell, PhD USDA, ARS, BHNRC Food Composition Lab Byrdwell@ba.ars.usda.gov http://www.Byrdwell.com

  35. You may find these Byrdwell.com Websites helpful: www.Sphingomyelin.com www.PhosphatidylCholines.com www.PhosphatidylEthanolamines.com www.PhosphatidylSerines.com www.PhosphatidylGlycerols.com www.PhosphatidylInositols.com www.Triacylglycerols.com

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