Lyso-form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids.

In this work we report the development of a novel methodology for the determination of stereospecificity of diacyl glycerophospholipids, including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols (PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized in negative ion mode. This methodology uses MS(2) recorded on a hybrid quadrupole time-of-flight mass spectrometer to determine the stereospecificity of diacyl glycerophospholipids based on the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2](-) ) and as ketenes ([M-(Sn2-H(2) O)](-) ) exhibited consistently higher intensity than their counterpart ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1](-) and [M-(Sn1-H(2) O)](-) ). Therefore, we concluded that an empirical fragmentation rule can be used to precisely determine the stereospecificity of diacyl glycerophospholipids, primarily on the basis of relative abundance of the lyso-form fragment ions. We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined. Combining the novel methodology reported in this work with the currently widely practiced mass spectrometric techniques such as multiple precursor ion scans (MPIS), fatty acyl scans (FAS), and multidimensional mass spectrometry based shotgun lipidomics (MDMS-SL), should enable a reliable and convenient platform for comprehensive glycerophospholipid profiling.