Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Abstract

SUMMARYQuantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC-MS) analysis. Proper peak annotation of the fatty acids in the LC-MS-based methods has been achieved by LC-MS measurements of authentic standard compounds and elemental formula annotation supported by 13 C isotope-labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC-MS and GC-FID of two previously characterized Arabidopsis thaliana knock-out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC-MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC-MS and GC-FID analyses are comparable, but that because of higher sensitivity and selectivity the LC-MS-based method allows for a broader coverage and determination of novel fatty acids.

Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample

“…In previous studies, we have analyzed the MTBE-derived lipid phase for the identification and analysis of lipid species from Arabidopsis thaliana dry seeds [48, 53], seedlings [32, 49], leaves [23, 27, 50–54], roots [23] and flowers [54]. Additionally, we applied the same method, with minor adaptations in the extraction process, to compare the lipid composition of mammalian tissues including brain, kidney and skeletal muscle of mice, rhesus macaques, chimpanzees and humans [58–60].…”

“…Furthermore, we applied the described method to determine the lipid composition of the green algal species Scenedesmus (Acutodesmus) obliquus [56] and the model fly Drosophila melanogaster [57]. Moreover, the method has been proven useful also for lipid profiling of 124 lipid species from the marine diatom ( Thalassiosira pseudonana ) [53, 61] and the biddulphioid diatom ( Biddulphia biddulphiana ) [61]. …”

Comprehensive metabolic reprograming in freshwater Nitzschia palea strains undergoing nitrogen starvation is likely associated with its ecological origin

“…From each lipid-containing non-polar phase an aliquot was dried in vacuum and saponification was performed according to [59] using methanol 6% KOH (w/v). This was followed by acidification with 29% HCl (v/v) and, finally, fatty acids were extracted with a chloroform/heptane mixture.…”

Mass spectrometry as a quantitative tool in plant metabolomics

“…Lipidomics has long been recognized as a subfield of the more comprehensive metabolomics research area, and therefore the extraction and analysis of the plant lipidome falls outside of the scope of this review. Detailed sensitive MS-based methods for the quantitative analysis of lipid-related species can also be found elsewhere [42,[71][72][73][74][75][76].…”