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Papers In Press, published online ahead of print August 1, 2009
J. Lipid Res., doi:10.1194/jlr.D800051-JLR200

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Journal of Lipid Research, Vol. 50, 1692-1707, August 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers^[S]

Rebecca L. Shaner^*, Jeremy C. Allegood^1,*, Hyejung Park, Elaine Wang, Samuel Kelly, Christopher A. Haynes, M. Cameron Sullards^*, and Alfred H. Merrill, Jr.^2,*,

^* Schools of Chemistry and Biochemistry, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0230
Biology, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0230

² To whom correspondence should be addressed. email: al.merrill{at}biology.gatech.edu

Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS³ protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. JLR LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.

Supplementary key words lipid maps • lipidomics internal standards • mass spectrometry • RAW 264.7 macrophage • sphingolipids

Abbreviations: CE, collision energies; Cer, ceramide; Cer1P, ceramide 1-phosphate; CXP, collision cell exit potentials; DHCer, dihydroceramide; DHCer1P, dihydroceramide 1-phosphate; DHSM, dihydrosphingomyelin; DP, declustering potential; EP, entrance potential; FBS, fetal bovine serum; FP, focusing potential; GalCer, galactosylceramide; GlcCer, glucosylceramide; HexCer, hexosylceramide; LC, liquid chromatographic; LacCer, lactosylceramide; LC-MS/MS, liquid chromatography tandem mass spectrometry; MRM, multiple reaction monitoring; PBS, phosphate-buffered saline; Q, quadrupole; QQQ, triple quadrupole; QTrap, quadrupole linear-ion trap; SM, sphingomyelin; Sa, sphinganine; Sa1P, sphinganine 1-phosphate; So, sphingosine; S1P, sphingosine 1-phosphate

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