Unveiling the compositional variety of cardiolipins in Rhodobacter sphaeroides by liquid chromatography with electrospray ionization and multistage collision-induced dissociation mass spectrometry.

Cardiolipins (CL) contained in the lipid extracts of the photosynthetic bacterium Rhodobacter sphaeroides (strain R26) were systematically characterized by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry, performed in single (MS), tandem (MS/MS) and sequential (MS) modes using a linear ion trap mass spectrometer. The total number of carbon atoms and C=C bonds of each CL and, subsequently, those related to each of the constituting phosphatidic acid (PA) units, along with the location of the latter on the central glycerol backbone, were inferred from MS and MS/MS data, respectively. Moreover, the composition and location of both acyl chains on the glycerol backbone of each PA unit was obtained by MS measurements, an approach used for the first time for the structural elucidation of CL in R. sphaeroides. As a result, an unprecedented profile of CL in this bacterium was obtained, with 27 main species characterized, many of which are represented by compositional or regiochemical isomers. Interestingly, such a variability is generated from a limited number of different acyl chains, either saturated (i.e. 12:0, 16:0, 17:0, 18:0, 19:0) or mono-unsaturated (16:1, 18:1). The absence of polyunsaturated chains, more susceptible to oxidation damage, appeared to be indirectly related to the lack of carotenoids potentially acting as antioxidant agents, a specific feature of R. sphaeroides R26. The occurrence of odd-numbered acyl chains was ascribed to the need to guarantee membrane fluidity, through a less compact packing of CL, thus compensating for the lack of CL bearing polyunsaturated side chains. Graphical abstract Representation of MS signals due to carboxylate anions that would be detected, as separate couples, in the fragmentation spectra of the anions resulting from the two phosphatidic acid units included in a cardiolipin molecule bearing four different acyl chains.