Analysis of artificially oxidized cardiolipins and monolyso-cardiolipins via liquid chromatography/high-resolution mass spectrometry and Kendrick mass defect plots after hydrophilic interaction liquid chromatography based sample preparation

Abstract

Rationale: Cardiolipins (CL) are a special lipid class which plays a main role in energy metabolism in mitochondria and is involved in apoptosis. In contrast to other glycerophospholipids, they contain four fatty acyl residues which results in a high structural diversity. Oxidation, for example by reactive oxygen species, or lyso forms such as monolyso-CL (MLCL), increases this diversity. Mass spectrometric analysis and computational identification of CL, MLCL and their oxidation products is therefore a challenging task.

Methods: In order to distinguish CL, MLCL and their oxidation products, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. A hydrophilic interaction liquid chromatography (HILIC)-based solid-phase extraction (SPE) clean-up approach was developed for CL enrichment. Graphical analysis of CL, MLCL and their oxidation products was carried out by a three-dimensional Kendrick mass defect (3D-KMD) plot module, as well as a refined lipid search module of the open-source metabolomics data mining software MZmine 2.

Results: The HILIC-based SPE clean-up enabled complete separation of polar and nonpolar lipid classes. A yeast (Saccharomyces cerevisiae) lipid extract, which was artificially oxidized by means of the Fenton reaction, was analyzed by the developed LC/MS/MS method. CL species with differences in chain length and degree of unsaturation have been separated by high-performance liquid chromatography (HPLC). In total 66 CL, MLCL and oxidized species have been identified utilizing 3D-KMD plots in combination with database matching using MZmine 2. For further characterization of annotated species, MS/MS experiments have been utilized.

Conclusions: 3D-KMD plots capturing chromatographic and high-resolution mass spectrometry data have been successfully used for graphical identification of CL, MLCL as well as their oxidized species. Therefore, we chose multiple KMD bases such as hydrogen and oxygen to visualize the degree of unsaturation and oxidation capturing chromatographic data by means of a color-coded paint scale as the third dimension. In combination with database matching, the analysis of low concentrated lipid species in complex samples has been significantly improved.