Analysis of in Vivo Levels of Acyl-Thioesters with Gas Chromatography/Mass Spectrometry of the Butylamide Derivative

The analysis of the in vivo level and composition of acyl-thioester pools, such as acyl-ACP, has been accomplished by selective formation of acyl-butylamides and subsequent analysis by gas chromatography/mass spectrometry. The acyl-butylamide derivative was synthesized by direct aminolysis with n-butylamine. The reaction was specific for thioester-linked acyl groups and 90% conversion was achieved with acyl-ACP and acyl-CoA in aqueous solution. Electron ionization mass spectra exhibited two intense diagnostic ions m/ z 115 and 128 common to butylamides of saturated and monounsaturated fatty acids. Mixtures of butylamides with fatty acid moieties ranging between C 4 and C 20 were analyzed by selective ion monitoring gas chromatography/mass spectrometry set to 115 and 128 amu. The limit for the quantitative analysis of the long-chain 18:0- and 18:1-butylamides was 1.5 pmol and the detection limit was less than 0.5 pmol. The utility of this method was demonstrated by analysis of two model systems: standard acyl-CoA mixtures and in vivo levels of spinach leaf acyl-ACP. A purification protocol based on DE 52 anion exchange chromatography was necessary in order to separate spinach acyl-ACP and acyl-CoA from tissue extracts. The acyl composition obtained from total spinach leaf acyl-ACP by selective ion monitoring gas chromatography/mass spectrometry of the butylamide derivatives matched with the direct analysis of the same sample by urea-polyacrylamide gel electrophoresis and subsequent scanning densitometry of the anti-spinach ACP immunoblot.