Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry.

Complete structural characterization of complex lipids, such as glycerophospholipids, by tandem mass spectrometry (MS/MS) continues to present a major challenge. Conventional activation methods do not generate fragmentation patterns that permit the simultaneous discernment of isomers which differ in both the positions of acyl chains on the glycerol backbone and the double bonds within the acyl chains. Herein we describe a hybrid collisional activation/UVPD workflow that yields near-complete structural information for glycerophospholipids. This hybrid MS(3) strategy affords the lipid's sum composition based on the accurate mass measured for the intact lipid as well as highly specific diagnostic product ions that reveal both the acyl chain assignment (i.e., sn-position) and the site-specific location of double bonds in the acyl chains. This approach is demonstrated to differentiate sn-positional and double-bond-positional isomers, such as the regioisomeric phosphatidylcholines PC 16:0/18:1(n-9) and PC 18:1(n-9)/16:0, and has been integrated into an LC-MS(3) workflow.