Analysis of phosphate and phosphate containing headgroups enzymatically cleaved from phospholipids of Bacillus subtilis by capillary electrophoresis.

A new, fast, selective, and reliable capillary electrophoresis method has been developed for analysis of selected phosphoesters (phosphoserine, phosphoethanolamine, phosphoglycerol) and phosphate. The method is based on separation of specific phosphate containing headgroups (phosphoesters) which are cleaved from the glycerol skeleton of a phospholipid by a regioselective enzyme (phospholipase C). Analysis of intact phospholipids with the same polar headgroup but different fatty acids shows that fatty acid composition has a high impact on separation of phospholipids, so analysis of separated polar headgroups, which avoids this influence, represents a much more suitable approach for phospholipid class research. Optimization of method parameters results in running buffers of relatively narrow pH interval (pH about 10) where all phosphoesters are separated. Further method validation has shown that direct UV detection has a sufficient detection limit for all analytes to perform suitable analyses of cell membrane lipids. The optimized method was tested on the lysate of cell membrane of Bacillus subtilis, where all analytes were determined.