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Journal of Lipid Research, Vol. 49, 1113-1125, May 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry^*

Christopher A. Haynes^*, Jeremy C. Allegood, Kacee Sims, Elaine W. Wang^*, M. Cameron Sullards^*, and Alfred H. Merrill, Jr.^1,*,

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

^* These studies were funded by National Institutes of Health Grant GM-069338 to the Lipid MAPS Consortium. The authors thank Dr. Walter Shaw and Avanti Polar Lipids for the provision of fatty acyl-CoA standards.

Published, JLR Papers in Press, February 20, 2008.

¹ To whom correspondence should be addressed. e-mail: al.merrill{at}biology.gatech.edu

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture (10⁶–10⁷), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 ± 1.0 pmol/10⁶ RAW264.7 cells vs. 80.4 ± 6.1 pmol/10⁶ MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.

Supplementary key words lipidomics • metabolomics • RAW264.7 • MCF7 • lignoceroyl-CoA • nervonoyl-CoA • cerotoyl-CoA

Abbreviations: CAD, collisionally activated dissociation; CE, collision energy; CoASH, coenzyme A; CXP, collision cell exit potential; DP, desolvation potential; LOQ, limit of quantitation; MRM, multiple reaction monitoring; TEA, triethylamine; TEAA, triethylammonium acetate

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