No abstract

This work shows how the addition of chromatographic information, i.e. the retention time, to a classical two-dimensional Kendrick mass plot enables rapid and accurate analysis of LC/HRMS datasets, exemplified on a green alga (C. reinhardtii) sample. Three-dimensional Kendrick mass plots have improved lipid class identification and fast spotting of falsely annotated lipid species.

In recent years, proprietary and open-source bioinformatics software tools have been developed for the identification of lipids in complex biological samples based on high-resolution mass spectrometry data. These existent software tools often rely on publicly available lipid databases, such as LIPID MAPS, which, in some cases, only contain a limited number of lipid species for a specific lipid class. Other software solutions implement their own lipid species databases, which are often confined regarding implemented lipid classes, such as phospholipids. To address these drawbacks, we provide an extension of the widely used open-source metabolomics software MZmine 2, which enables the annotation of detected chromatographic features as lipid species. The extension is designed for straightforward generation of a custom database for selected lipid classes. Furthermore, each lipid's sum formula of the created database can be rapidly modified to search for derivatization products, oxidation products, in-source fragments, or adducts. The versatility will be exemplified by a liquid chromatography−high resolution mass spectrometry data set with postcolumn Paterno−Buchi derivatization. The derivatization reaction was performed to pinpoint the double bond positions in diacylglyceryltrimethylhomoserine lipid species in a lipid extract of a green algae (Chlamydomonas reinhardtii) sample. The developed Lipid Search module extension of MZmine 2 supports the identification of lipids as far as double bond position level.

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.

Anterograde transport of late endosomes or lysosomes (LE/Lys) is crucial for proper axon growth. However, the role of energetic nutrients has been poorly explored. Malonyl-CoA is a precursor of fatty acids, and its intracellular levels highly fluctuate depending on glucose availability or the energy sensor AMP-activated protein kinase (AMPK). We demonstrate in HeLa cells that carnitine palmitoyltransferase 1C (CPT1C) senses malonyl-CoA and enhances LE/Lys anterograde transport by interacting with the endoplasmic reticulum protein protrudin and facilitating the transfer of Kinesin-1 from protrudin to LE/Lys. In cultured mouse cortical neurons, glucose deprivation, pharmacological activation of AMPK or inhibition of malonyl-CoA synthesis decreases LE/Lys abundance at the axon terminal, and shortens axon length in a CPT1C-dependent manner. These results identify CPT1C as a new regulator of anterograde LE/Lys transport in response to malonyl-CoA changes, and give insight into how axon growth is controlled by nutrients.