Quantitation of fatty acyl-coenzyme A molecular species in mammalian cell extracts by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS)

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

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332

Corresponding Author: gtg983j{at}mail.gatech.edu

Fatty acyl-coenzyme As (fatty acyl-CoAs) participate in numerous cellular processes and have been analyzed by many approaches although none have been applied to a wide range of species in relatively small samples, such as cells in culture. This manuscript describes a method for quantitation of subpicomole amounts of long- and very-long-chain fatty acyl-CoAs by reverse phase liquid chromatography combined with electrospray ionization tandem mass spectrometry using multiple reaction monitoring of precursor and product ion pairs in positive ion mode. Odd-chain length fatty acyl-CoAs are used as internal standards for quantitation after biological samples have been ascertained to contain insignificant amounts of these species. The 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 to 10⁷), with analyses of RAW264.7 cells (a mouse macrophage line) and MCF7 cells (a human breast carcinoma line) given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (i.e., 12 + 1.0 pmol / 10⁶ RAW264.7 cells versus 80.4 + 6.1 pmol / 10⁶ MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths = C20 comprise < 5% 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 fairly simple and robust method should facilitate inclusion of this family of compounds in a wide range of studies, including “lipidomics” and “metabolomics.”

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