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Comprehensive Quantitative Analysis of Bioactive Sphingolipids by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

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Lipidomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 579))

Summary

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1P) and this has necessitated the development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), ceramides (Cers), ceramide 1-phosphates (Cer-1P), glucosyl/galactosyl-ceramides (Glu-Cers), and sphingomyelins (SMs) is performed on a Thermo Fisher Scientific triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues, or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a LC/MS/MS system. Qualitative analysis (identification) of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target analyte, synthetic standards, and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration, using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit of subpicomole level. This methodology constitutes a “Lipidomic” approach to study the SPLs metabolism, defining a function of distinct subspecies of individual bioactive SPL classes.

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References

  1. Sastry, P.S., (1985) Lipids of nervous tissue: composition and metabolism. Prog Lipid Res 24(2), 69–176

    Article  PubMed  CAS  Google Scholar 

  2. Vos, J.P., Lopes-Cardozo, M., Gadella, B.M., (1994) Metabolic and functional aspects of sulfogalactolipids. Biochim. Biophys. Acta 1211, 125–149

    Article  PubMed  CAS  Google Scholar 

  3. Hannun, Y.A., Obeid, L.M., (2002) The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 277, 25847–25850.

    Article  PubMed  CAS  Google Scholar 

  4. Adams, J., Ann, Q., (1993) Structure determination of sphingolipids by mass spectrometry. Mass Spectrom. Rev. 12, 51–85.

    CAS  Google Scholar 

  5. Ann, Q., Adams, J., (1993) Structure-specific collision-induced fragmentation of ceramides cationized with alkali-metal ions. Anal. Chem. 22, 7–13.

    Article  Google Scholar 

  6. Ann, Q., Adams, J., (1993) Collision-induced decomposition of sphingomyelins for structural elucidation. Biol. Mass Spectrom. 22, 285–294.

    CAS  Google Scholar 

  7. Sullards, M.C., (2000) Sphingolipid metabolism and cell signaling. Methods Enzymol 312, 32–45.

    Article  PubMed  CAS  Google Scholar 

  8. Gu, M., Kerwin, J.L., Watts, J.D., Aebersold, R., (1997) Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry. Anal. Biochem. 244, 347–356.

    CAS  Google Scholar 

  9. Mano, N., Oda, Y., Yamada, K., Asakawa, N., Katayama, K., (1997) Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry. Anal. Biochem. 244, 291–300.

    CAS  Google Scholar 

  10. Liebisch, G., Derobnik, W., Reil, M., Trumbach, B.R., Arnecke, R., Olgemoller, B., Roscher, A., Schmitz, G.J., (1999) Quantitative measurement of different ceramide spiecies from crude cellular lipid extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Lipid Res. 40, 1539–1546.

    CAS  Google Scholar 

  11. Sullard, M.C., Merrill, A.H., (2001) Analysis of sphingosine-1-phosphate, ceramides and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Science’s stke. 67, 1–11.

    Google Scholar 

  12. Folch, J., Lees, M., Sloane, H.S., (1956) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 196, 497–509.

    Google Scholar 

  13. Bligh, E.G., Dyer, W.J., (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.

    Article  CAS  Google Scholar 

  14. Bielawski, J., Szulc, Z.M., Hannun, Y.A., Bielawska, A., (2006) Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods 39, 82–91.

    Article  PubMed  CAS  Google Scholar 

  15. Bodennec, J., Brichon, G., Zwingelstein, G., Portoukalian, J., (2000) Purification of sphingoid classes by solid-phase extraction with aminopropyl and weak cation exchange cartridges. Methods Enzymol.. 312, 101–114.

    Article  PubMed  CAS  Google Scholar 

  16. Kralik, S.F., Du, X., Patel, C., Walsh, J.P., (2001) A method for quantitative extraction of sphingosine 1-phosphate into organic solvent. Anal. Biochem. 294, 190–193.

    CAS  Google Scholar 

  17. Ali, M.T., David, B.D., Peter, S.H., Valerie Boote Oral Microbiology Laboratory, (1998) Eukaryotic cell signaling and transcriptional activation induced by bacterial porins. FEMS Immunol. Medical Microbiol. 21(1), 57–64

    Article  Google Scholar 

  18. Korachi, M., Blinkhorn, A.S., Drucker, D.B. (2002) Analysis of phospholipid molecular species distributions by fast atom bombardment mass spectrometry (FAB-MS). Eur. J. Lipid Sci. Technol. 104, 50–56

    Article  CAS  Google Scholar 

  19. Murray, K.E., Schulten, H.R., (1981) Field desorption mass spectrometry of lipids. I. The application of field desorption mass spectrometry to the investigation of natural waxes. Chem. Phys. Lipids 29, 11–21.

    Article  CAS  Google Scholar 

  20. Ma, Y.-C., and Kim, H.-Y. (1995) Development of the on-line HPLC/thermospray MS method for the analysis of phospholipid molecular species in brain. Anal. Biochem. 226, 293–301.

    Article  PubMed  CAS  Google Scholar 

  21. Edmond de, H., (1996) Tandem mass spectrometry: a primer. J. Mass Spectrom. 31, 129–137

    Article  Google Scholar 

  22. Jackson, S.N., Wang, H.Y.J., Woods, A.S. (2005) Direct tissue analysis of phospholipids in rat brain using MALDI-TOFMS and MALDI-ion mobility-TOFMS. J. Am. Soc. Mass Spectrom. 16, 133–138

    Article  PubMed  CAS  Google Scholar 

  23. Suzuki, Y., Suzuki, M., Ito, E., Goto-Inoue, N., Miseki, K., Iida, J., Yamazaki, Y., Yamada, M., Suzuki, A., (2006) Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased lase power and collision gas flow. J. Biochem. 139, 771–777.

    Article  CAS  Google Scholar 

  24. Ugarov, M., Egan, T., Koomen, J., Gillig, K.J., Fuhrer, K., Gonin, M., Schultz, J.A., (2004) Lipid/peptide/nucleotide separation with MALDI-Ion Momility-TOF MS. Anal. Chem. 76, 2187–2195.

    Google Scholar 

  25. Adams, J., Ann, Q., (1992) Structure determination of ceramides and neutral glycosphingolipids by collisional activation of (M + Li)+ ions. J. Am. Soc. Mass Spectrom. 3, 260–263.

    Article  Google Scholar 

  26. Vieu, C., Chevy, F., Rolland, C., Barbaras, R., Chap, H., Wolf, C., Perret, B., Collet, X., (2002) Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography. J. Lipid Res. 43, 510–522

    PubMed  CAS  Google Scholar 

  27. Isaac, G., Bylund, D., Masson, J.E., Markides, K.E., Bergquist, J., (2003) Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry. J. Neurosci. Method 128, 111–119.

    Article  Google Scholar 

  28. Karlsson, A.A., Michelsen, P., and Odham, G., (1998) Molecular species of sphingomyelin: Determination by high-performance liquid chromatography mass spectrometry with electrospray and high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization. J. Mass Spectrom. 33, 1192–1198.

    Article  PubMed  CAS  Google Scholar 

  29. Monick, M.M., Mallampalli, R.K., Bradford, M., McCoy, D., Gross, T.J., Flaherty, D.M., Powers, L.S., Cameron, K., Kelly, S., Merrill, A.H., Hunninghake, G.W., (2004) Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high level of acid ceramidase activity. J. Immunol. 173, 123–135.

    PubMed  CAS  Google Scholar 

  30. Adams, J.M., Pratipanawatr, T., Berria, R., Wang, E., DeFronzo, R.A., Sullard, M.C., Mandarino, L., (2004) Ceramide content is increased in skeletal muscle from obese insulin-resistant humans. Diabetes 53, 25–31.

    Article  PubMed  CAS  Google Scholar 

  31. Zheng, W., Kollmeyer, J., Symolon, H., Momin, A., Munter, E., Wang, E., Kelly, S., Allegood, J.C., Liu, Y., Peng, Q., Ramaraju, H., Sullard, M.C., Cobot, M., Merrill, A.H., (2006) Ceramides and other bioactive sphingolipids backbone in health and disease: Lipidomic analysis, metabolism and roles in memebrane structure, dynamic, signalic and autopaphy. Biochim. Biophys. Acta. 1758 (12), 1864–1884.

    Article  CAS  Google Scholar 

  32. Maceyka, M., Sankala, H., Hait, N.C., LeStunff, H., Liu, H., Toman, R., Collier, C., Zhang, M., Satin, L.S., Merrill, A.H., Milstien, S., Spiegel, S., (2005) SphK1 and SphK2, sphingosine kinase isoenzymes with opposition functions in sphingolipid metabolism. J. Biol. Chem. 280, 37118–37129.

    Article  PubMed  CAS  Google Scholar 

  33. Bose, R., Verheij, R., Haimovitz-Friedman, A., Scotto, K., Fuks, Z., Kolesnick, R.N., (1995) Ceramide synthase mediates daunorubicin-induced apoptosis; an alternative mechanism for generating death signals. Cells 82, 405–414.

    Article  CAS  Google Scholar 

  34. Luberto, C., Hannun, Y.A., (1998) Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation – Does sphingomyelin synthase account for the putative, phosphatidylcholine-specific phospholipase C? J. Biol. Chem. 273, 14550–14559.

    Article  CAS  Google Scholar 

  35. Bielawska, A., Perry, D.K., Hannun, Y.A., (2001) Determination of ceramides and diglycerides by the diglyceride kinase assay. Anal. Biochem. 298, 141–150.

    CAS  Google Scholar 

  36. Sullard, M.C., Wang, E., Peng, Q., Merrill, Jr. A.H., (2003) Metabolomic profiling of sphingolipids in human glioma Cell lines by liquid chromatography tandem mass spectrometry. Cell. Mol. Biol. 49, 789–797.

    Google Scholar 

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Acknowledgments

Financial support was provided by NCI Grant No. IPO1CA097132 and NIH/NCRR SC COBRE grant No. P20 RR017677. Special acknowledgement is for NCRR Grant No. CO6RR018823 providing laboratory space for Lipidomics Shared Resource in the CRI building of MUSC.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Lipidomic Core Mass Spectrometry Lab, Medical University of South Carolina, Charleston, SC, USA

    Jacek Bielawski, Jason S. Pierce, Justin Snider, Barbara Rembiesa, Zdzislaw M. Szulc & Alicja Bielawska

Authors
  1. Jacek Bielawski
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  2. Jason S. Pierce
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  3. Justin Snider
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  4. Barbara Rembiesa
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  5. Zdzislaw M. Szulc
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  6. Alicja Bielawska
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Editors and Affiliations

  1. Golden Leaf Rd. 215, Mars Hill, 28754, U.S.A.

    Donald Armstrong

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Bielawski, J., Pierce, J.S., Snider, J., Rembiesa, B., Szulc, Z.M., Bielawska, A. (2009). Comprehensive Quantitative Analysis of Bioactive Sphingolipids by High-Performance Liquid Chromatography–Tandem Mass Spectrometry. In: Armstrong, D. (eds) Lipidomics. Methods in Molecular Biology, vol 579. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-322-0_22

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  • DOI: https://doi.org/10.1007/978-1-60761-322-0_22

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-321-3

  • Online ISBN: 978-1-60761-322-0

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