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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sastry, P.S., (1985) Lipids of nervous tissue: composition and metabolism. Prog Lipid Res 24(2), 69–176
Vos, J.P., Lopes-Cardozo, M., Gadella, B.M., (1994) Metabolic and functional aspects of sulfogalactolipids. Biochim. Biophys. Acta 1211, 125–149
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.
Adams, J., Ann, Q., (1993) Structure determination of sphingolipids by mass spectrometry. Mass Spectrom. Rev. 12, 51–85.
Ann, Q., Adams, J., (1993) Structure-specific collision-induced fragmentation of ceramides cationized with alkali-metal ions. Anal. Chem. 22, 7–13.
Ann, Q., Adams, J., (1993) Collision-induced decomposition of sphingomyelins for structural elucidation. Biol. Mass Spectrom. 22, 285–294.
Sullards, M.C., (2000) Sphingolipid metabolism and cell signaling. Methods Enzymol 312, 32–45.
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.
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.
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.
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.
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.
Bligh, E.G., Dyer, W.J., (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.
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.
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.
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.
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
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
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.
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.
Edmond de, H., (1996) Tandem mass spectrometry: a primer. J. Mass Spectrom. 31, 129–137
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
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
Bielawska, A., Perry, D.K., Hannun, Y.A., (2001) Determination of ceramides and diglycerides by the diglyceride kinase assay. Anal. Biochem. 298, 141–150.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-60761-322-0_22
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-321-3
Online ISBN: 978-1-60761-322-0
eBook Packages: Springer Protocols