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). The molecular structure of phytosphingosine was confirmed by negative-ion LC-MS-MS, which also served as a rapid tool for screening the plant spingolipid desaturase activity with 2-min run time under multiple-reaction monitoring (MRM) mode. The C=C bond location of dehydrophytosphingosine was further identified by GC-MS after being converted into picolinyl derivatives. This assay combines multiple chromatographic and mass spectrometric techniques with gentle chemical procedures to provide capacities for rapid determination of the plant sphingolipid desaturase activity as well as identification of their active sites in the backbone of the sphingolipid species in yeast.