Structural Characterization of Unsaturated Glycerophospholipids by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

Structural elucidation of glycerophospholipids (GPLs), including the polar head group, the position of double-bond(s) along the fatty acyl substituents, and the positions of acyl groups on the glycerol backbone, using multiple-stage liner ion-trap (LIT) mass spectrometric approach is described in this paper. While the product-ion spectra from MSⁿ (n = 2, 3) on the [M + Li]⁺ or [M - H + 2Li]⁺ ions of GPL are readily applicable for discerning the phospholipid classes and for identifying and locating the fatty acid substituents on the glycerol backbone, the structural information from further dissociation of the dilithiated fatty acid cations produced from MSⁿ (n = 3, 4) on the [M - H + 2Li]⁺ ion of GPLs, as well as from further dissociation of the monolithiated fragment ion that bears the unsaturated fatty acid moiety produced from subsequent MSⁿ (n = 3,4) on the [M + Li]⁺ ions of GPLs, affords assignment of the position of double-bond(s) along the fatty acyl groups. The application of the present method in the structural characterization of GPL molecules from the lipid extracts of biological origin, including mixtures of phosphatidylglycerol and of phosphatidylserine without prior chromatographic separation, is also demonstrated. Since lithiated molecular species of GPL are readily formed by ESI, this multiple-stage LIT mass spectrometric approach provides a direct means for the near-complete structural characterization of all the GPLs, including the molecules in the lysophospholipid and plasmalogen subclasses.