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

RationaleCardiolipins (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.MethodsIn 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.ResultsThe 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.Conclusions3D‐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.

Analytical approaches for studying oxygenated lipids in the search of potential biomarkers by LC-MS

“…An alternative to overcome this issue is online-SPE, which allows the automatized lipid extraction before analysis in the LC (entry 42) [64]. C8 and C18-based SPE cartridges are amongst the most employed (entry 43) [65] although HILIC-based have been used for OxCLs [94]. Meanwhile, solid phase microextraction (SPME) employs sorbent-coated fibers where lipids migrate and diffuse directly from a headspace or liquid to the fiber, eliminating the washing step necessary for SPE.…”

“…Interestingly, in the case of OxCLs, some authors have conducted a classical high-performance thin-layer chromatography (HPTLC)phosphorus analysis to decrease the matrix effect, previous to RPLC-MS analysis (entry 35) [24]. Another attractive strategy to increase selectivity is 2D-LC/MS online analysis, which uses HILIC in the first dimension to eliminate lipid interferences followed by an automatic analyte transfer to perform RPLC separation as the second main dimension [94,110]. The online coupling is faster and prevents sample overhandling, avoiding undesired products.…”

“…From these, 8 oxidized glycerophosphatidylcholines (OxPCs) species containing a fragmented, oxidized short-chain fatty acid remnant at the sn-2 position, are available: (i) C4 truncated ( 16 To address this dearth of standards several approaches rely on in-house prepared mixtures of oxidized species generated in vitro, from non-oxidized commercial ones, (entries 17, 18, 26, 27) [17,18,26,27], as has been reviewed recently [119]. Thus, PAPC, SAPC, PLPC, and SAPE among others, and commercial TMCL, TLCL or bovine heart CL (BHCL) (with different content in TLCL depending on the provider), have been widely used to carry out different oxidation procedures, Table 2.2 [27,56,43,94,120]. Good examples are photo-oxidation (UVA irradiation) (entries 26) [51,55], airexposure oxidation (entry 18) [44], or under free radical conditions through already established methods such as the Fenton reaction (Fe(II)/H 2 O 2 ) for GPs (entries 17, 27) [27].…”

“…However, so far it was rather used based on the hydrogen KMD(H) to visualize the degree of unsaturation, but nowadays, visualization based on the oxygen KMD(O) allows to track the oxidation product. Such strategy was used for annotation of OxCLs and monolyso-CLs [94]. Multiple 3D-KMD are built by plotting horizontally KMD(H) against vertical KMD(O), and RT as third dimension represented through colorcoded heat map.…”

Epilipidomics platform for holistic profiling of oxidized complex lipids in blood plasma of obese individuals

“…C13-OOH < C9-OOH). Furthermore, previously introduced retention time (RT) mapping of oxidized lipids using Kendrick mass defect (KMD) plots 34 was employed here for visualization and manual inspection of the defined elution order to increase accuracy of the annotations in high-throughput epilipidome profiling experiments (Figure 3B). Importantly, using KMD vs RT plots we were able to spot previously unconsidered in-source fragmentation (ISF) of oxidized lipids.…”