Characterization of Cardiolipin as the Sodiated Ions by Positive-Ion Electrospray Ionization with Multiple Stage Quadrupole Ion-Trap Mass Spectrometry

The application of multiple-stage ion-trap (IT) mass spectrometric methods for the structural characterization of cardiolipin (CL), a 1,3-bisphosphatidyl-sn-glycerol that consists of four fatty acyl chains and three glycerol backbones (designated as A, B, and central glycerol, respectively), as the sodiated adduct ions in the positive-ion mode was evaluated. Following collisionally activated dissociation (CAD), the [M - 2H + 3Na]⁺ ions of CL yield two prominent fragment ion pairs that consist of the phosphatidyl moieties attached to the 1′- and 3′-position of the central glycerol, respectively, resulting from the differential losses of the diacylglycerol moieties containing A and B glycerol, respectively. The results are consistent with those previously described for the [M - H]^- and [M - 2H + Na]^- ions in the negative-ion mode, thus permitting assignment of the two phosphatidyl moieties attached to the 1′- or 3′-position of the central glycerol. The identities of the fatty acyl substituents and their positions on the glycerol backbones (glycerol A and B) are deduced from further degradation of the above ion pairs that give the fragment ions reflecting the fatty acid substituents at the sn-1 (or sn-1′) and sn-2 (or sn-2′) positions. The ions that arise from losses of the fatty acid substituents at sn-1 and sn-1′, respectively, are prominent, but the analogous ions from losses of the fatty acid substituents at sn-2 and sn-2′, respectively, are of low abundance in the MS² product-ion spectra. This feature further confirms the assignment of the positions of the fatty acid substituents. The similar IT multiple-stage mass spectrometric approaches including MS² and MS³ for structural characterization of CL using its [M + Na]⁺ and the [M - H + 2Na]⁺ ions are also readily applicable. However, their uses for structural characterization are less desirable because formation of the [M + Na]⁺ and the [M - H + 2Na]⁺ ions for CL is not predictable.