Analysis of ether glycerophosphocholines at the level of C=C locations from human plasma

Plasmanyl and plasmenyl glycerophosphocholine are ether lipids featuring the 1-O-alkyl or 1-O-alk-1′-enyl ether linkage at the sn-1 position of the glycerol backbone, respectively. Aberrant levels of ether glycerophosphocholines (ether PCs) have been correlated with cellular dysfunctions and various human diseases. Profiling ether PCs with accurate structural information is challenging because of the common presence of isomeric and isobaric species in a lipidome. The Paternò–Büchi (PB) reaction, a double bond (C=C) specific derivatization method, is capable of pinpointing C=C locations in unsaturated lipids, when coupled with subsequent tandem mass spectrometry (MS/MS). In this study, we have tailored the acetone PB reaction for the analysis of ether PCs. PB-MS/MS via low energy collision-induced dissociation (CID) provides diagnostic ions specific to the alkenyl ether C=C bond, which are different from those derived from the isolated C=C bond in the alkyl or acyl chain, thereby facilitating the distinction of isomeric plasmenyl from plasmanyl PCs. PB-MS/MS coupled with high resolution MS and multi-stage MS/MS further enable confident identification of isomeric ether PCs and isobaric diacyl PCs from mixtures. A total of 45 ether PCs in human plasma have been identified for ether linkage type and chain composition, while 28 ether PCs have structures being fully characterized down to C=C locations.