Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry.
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 ( approximately 10(6)-10(7)), 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/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) 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.
Haynes
Christopher A
CA
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA.
Allegood
Jeremy C
JC
Sims
Kacee
K
Wang
Elaine W
EW
Sullards
M Cameron
MC
Merrill
Alfred H
AH
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007700:Kinetics; D008264:Macrophages; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
D000214:Acyl Coenzyme A