Identification of plant sphingolipid desaturases using chromatography and mass spectrometry.

This manuscript describes an efficient analytical assay combining high-performance liquid chromatography with UV detection (HPLC-UV), liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS), and gas chromatography with mass spectrometry (GC-MS) for the characterization and C=C bond localization on the long chain base of sphingolipids in yeast extracts in order to identify the plant sphingolipid desaturases activity. Samples of wild type control and transgenic yeast expressing putative sphingolipid desaturases were hydrolyzed into long chain bases. Mono-unsaturated long chain base, dehydrophytosphingosine (t18:1), in transgenic yeast as a result of the function of plant sphingolipid desaturase was detected with cis, trans-isomers resolution by reverse phase HPLC-UV as DNP (2,4-dinitrophenyl) derivatives along with saturated phytosphingosine (t18:0).