Toward total structural analysis of cardiolipins: Multiple-stage linear ion-trap mass spectrometry on the [M - 2H + 3Li]⁺ ions

ESI multiple-stage linear ion-trap (LIT) mass spectrometric approaches for a near-complete structural characterization of cardiolipins (CLs), including identification of the fatty acyl substituents, assignment of the fatty acid substituents on the glycerol backbone, and location of the double-bond(s) or cyclopropyl group along the fatty acid chain are described. Upon collisionally activated dissociation (CAD) on the [M - 2H + 3Li]⁺ ions of CL in an ion-trap (MS²), two sets of fragment ions (designated as (a + 136) and (b + 136) ions) analogous to those previously reported for the [M - 2H + 3Na]⁺ ions were observed, leading to assignment of the phosphatidyl moieties attached to 1'- or 3'-position of the central glycerol. Further dissociation of the (a + 136) (or (b + 136)) ions (MS³) gives rise to the (a + 136 - R_{1(or 2)}CO₂Li) (or b + 136 - R_{1(or 2)}CO₂Li) ion pairs that identify the fatty acid moieties and their position on the glycerol backbone. This is followed by MS⁴ on the (a + 136 - R_{1(or 2)}CO₂Li) (or b + 136 - R_{1(or 2)}CO₂Li) ion to eliminate a tricylic glycerophosphate ester residue (136 Da) to yield the (a - R_{1(or 2)}CO₂Li) ion, which is then subjected to MS⁵. The MS⁵ spectrum contains the structural information that locates the double-bond(s) or cyclopropyl group of the fatty acid substituents. Finally, the subsequent MS⁶ on the dilithiated fatty acid ions generated from MS⁵ also yields feature ions that confirm the assignment.