Differentiation of 1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl Glycerophospholipids by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

We described linear ion-trap mass spectrometric approaches applying MS³ and MS⁴ toward to the structural characterization of 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl-, and diacyl-glycerophospholipids (GPL) as the [M - H]^- ions desorbed by ESI in negative-ion mode. Further dissociation of the [lM - H - R₂CO₂H - polar head group]^- ions from the [M - H]^- ions of GPL that have undergone the consecutive losses of the fatty acid substituent at sn-2 and the polar head group readily gives the structural information of the radyl group at sn-1, resulting in structural differentiation among the 1-O-alk-1′-enyl-2-acyl-, 1-O-alkyl-2-acyl, and diacyl-glycerolphospholipid molecules. The distinction between a 1-O-alk-1′-enyl-2-acyl- and a 1-O-alkyl-2-acyl-GPL is based on the findings that the MS³ (or MS⁴) spectrum of the [M - H - R₂CO₂H - polar head group]^- ion from the former compound is dominated by the alkenoxide anion that represents the radyl moiety at sn-1, while the spectrum from the latter compound is dominated by the ion at m/z 135 arising from further loss of the 1-O-alkyl group as an alcohol. Another important notion is that the optimal collision energy required for acquiring the former spectrum is significantly lower than that required for obtaining the latter spectrum. Using the approaches, we are able to reveal the structures of several isobaric isomers in GPL mixtures of biological origin. Because the [M - H]^- ions are readily formed by various GPL classes (except glycerophosphocholine) in the negative-ion mode, these mass spectrometric approaches should have broad application in the structural identification of GPLs.