Molecular species analysis of arachidonate containing glycerophosphocholines by tandem mass spectrometry

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Abstract
Carboxylate anions arising from collision-induced dissociation (CID) of the [M - 15]- ion produced by fast atom bombardment (FAB) of glycerophosphocholine (GPCho) were previously shown to be produced in an abundance ratio of 1:3 for the carboxylic acids esterified at sn - 1 an sn - 2, respectively. This observation has been confirmed in a series of 13 synthetic GPCho molecular species. A good correlation was found between the isomeric purity of GPCho molecular species as determined by negative-ion FAB/CID analysis and the isomeric purity of the sn - 2 fatty acid using a phospholipase A2 assay. Negative-ion FAB mass spectra of several 1-OMICRON-alkyl-2-acyl-GPCho molecular species were found to be similar to those of diacyl GPCho. However, the CID spectra from the major high-mass ions are different from those of the diacyl species in that the [M - 15]- ion yields only one carboxylate anion and the [M - 86]- undergoes a neutral loss of the sn - 2 carboxylic acid as a major decomposition product. These results suggest several rules useful for structural characterization of GPCho molecular species by negative-ion tandem mass spectrometry (MS/MS): (1) For diacyl species, the mass of the two carboxyl anions plus the mass of the GPCho backbone (minus a methyl group) must correspond to the mass of the [M - 15] anion; (2) for diacyl species there is a carboxylate anion ratio approximately 1:3 for the substituents at sn - 1 and sn - 2; and (3) for alkylether species, only one fatty acyl group is present, and the difference between the ]M - 15[ ion and the GPCho backbone (minus methyl) plus the fatty acyl group at sn - 2 corresponds to an alkylether substituent. (4) Assignment of ether-linked molecular species can be made from the [M - 86]- ion, which has a strong neutral loss of th sn - 2 fatty acid. Analysis of GPCho isolated from human neutrophils by total lipid extraction and normal-phase HPLC was carried out by negative-ion FAB and MS/MS. The major arachidonate-containing molecular species, which comprise only 5% of total GPCho, were identified by using precursor ion scans for the arachidonate anion, m/z 303. Decomposition of identified precursor ions permitted the assignment of those molecular species of GPCho that contain arachidonate at sn - 2 and identification of the substituent at the sn - 1 position. These results were compared to previously identified molecular species from human neutrophils. Several minor arachidonate-containing molecular species were tentatively identified.