1. Division of Bioorganic Chemistry and Molecular Pharmacology Department of Medicine Washington University School of Medicine - St. Louis Shotgun Lipidomics of Cardiolipin Molecular Species Xianlin Han, Kui Yang, Jingyue Yang, Hua Cheng, and Richard W. Gross

2. Cellular Lipid Extract(s) of a Biological Sample Individual Molecular Species of Lipids ESI/MS Shotgun Lipidomics What is shotgun lipidomics?

3. Shotgun lipidomics includes: Multiplexed extractions Intrasource separation and selective ionization Multi-dimensional MS identification Quantitation using a two-step procedure Bioinformatics and data processing J. Lipid Res. 44 (2003), 1071-1079; Mass Spectrom. Rev. 24 (2005), 367-412; Expert Rev. Proteomics 2 (2005) 253-264

4. Shotgun lipidomics for analyses of cardiolipin molecular species Identification and Quantitation (Applications) J. Lipid Res. 47 (2006), 864

5. Shotgun lipidomics for analyses of cardiolipin molecular species Identification and Quantitation (Applications) Hsu, F.F., et al. (2005) J. Am. Soc. Mass Spectrom. 16, 491; Valianpour, F. (2002) Clin. Chem. 48, 1390; Sparagna, G.C. (2005) J. Lipid Res. 46, 1196. J. Lipid Res. 47 (2006), 864

6. What is cardiolipin? One minor phospholipid class, almost exclusively present in mitochondria; Two phosphates; Three glycerols; Four identical/almost identical acyl chains predominant in most of mammalian tissues.

7. Cardiolipin is a class of very important phospholipids: Highly anionic: interact with different cations and proteins, and influence ion channel function Large aliphatic chain to polar head group volume: promote membrane fusion and fission Specific binding with proteins in ETC; Anchoring cytochrome c (apoptosis) Barth’s syndrome (altered cardiolipin profile) ……

8. Identification

9. Doubly charged chemical nature ESI mass spectrometers with modest to high mass resolution (both QqQ- and QqTOF type) Identification: The basics of the technique

10. Identification: Negative-ion ESI/MS analysis of a lipid extract of mouse heart by direct infusion utilizing a QqQ-type instrument (FWHM 0.3 Th)

12. Identification: Negative-ion ESI/MS analysis of a lipid extract of mouse heart by direct infusion utilizing a QqTOF-type instrument

13. Identification: Product-ion analysis of cardiolipin molecular species in mouse heart lipid extract utilizing a QqQ-type instrument (FWHM 0.3 Th)

14. Identification: 2D MS analysis of some representative building blocks of mouse heart cardiolipin molecular species after direct infusion utilizing a QqQ-type instrument (FWHM 0.3 Th)

15. Quantitation

16. Quantitation: Quantitative analyses of different molar ratio mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqQ-type instrument Dynamic range of quantitation Linear correlation

17. Quantitation: Quantitative analyses of equimolar mixtures of T14:0 and T18:1 cardiolipin molecular species utilizing a QqTOF-type instrument

20. Quantitation: De-isotoping of cardiolipin molecular species based on [M+1] 2- isotopomer intensity (area)

21. IsotopomersIsotope RatioPeak Intensity (area) M192.42 I 1 /n M+10.01082nI 1 M+20.01082 2 n(n-1)/20.00541(n-1)I 1 ……………… Quantitation: De-isotoping of cardiolipin molecular species based on [M+1] 2- isotopomer intensity (area)

22. IsotopomersIsotope RatioPeak Intensity (area) M192.42 I 1 /n M+10.01082nI 1 M+20.01082 2 n(n-1)/20.00541(n-1)I 1 ……………… De-isotoping based on [M+1] 2- peak intensity (area): I total =I 1 x [92.42/n +1 + 0.00541(n - 1) + 1.95x10 -5 (n -1)(n - 2) + ……] Quantitation: De-isotoping of cardiolipin molecular species based on [M+1] 2- isotopomer intensity (area)

23. Quantitation: The effects of “ion suppression” on cardiolipin quantitation by shotgun lipidomics

24. Applications

25. Application: Shotgun lipidomics analyses of mouse skeletal muscle cardiolipin molecular species

26. Conclusion: Cardiolipin molecular species can be “recognized” by searching for the doubly-charged ions Cardiolipin molecular species can be identified by the specific neutral loss of ketenes from the doubly-charged molecular ions Cardiolipin molecular species can be quantitated after de- isotoping based on [M+1] 2- isotopomer intensity (area) Quantitation of cardiolipin can be performed using both QqQ- and QqTOF-type instruments with care “Ion suppression” does not affect quantitation of cardiolipin at the low concentration region using shotgun lipidomics