A combined flow injection/reversed phase chromatography – high resolution mass spectrometry workflow for accurate absolute lipid quantification with¹³C- internal standards

Abstract

We propose a fully automated novel workflow for lipidomics based on flow injection- followed by liquid chromatography high resolution mass spectrometry (FI/LC-HRMS). The workflow combined in-depth characterization of the lipidome achieved via reversed phase LC-HRMS with absolute quantification as obtained by a high number of lipid species-specific- and/or retention time (RT) matched/class-specific calibrants. The lipidome of 13C labelled yeast (LILY) provided a cost efficient, large panel of internal standards covering triacylglycerols (TG), steryl esters (SE), free fatty acids (FA), diacylglycerols (DG), sterols (ST), ceramides (Cer), hexosyl ceramides (HexCer), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), cardiolipins (CL), phosphatidylinositols (PI), phosphatidylserines (PS), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). In order to exploit the full potential of isotopically enriched biomass, LILY was absolutely quantified on demand via reversed isotope dilution analysis using FI-HRMS. Subsequent LC-HRMS analysis integrated different calibration strategies including lipid species-specific standards for >90 lipids. Extensive measures on quality control allowed to rank the calibration strategies and to automatically selected the calibration strategy of highest metrological order for the respective lipid species. Overall, the workflow enabled a streamlined analysis pipeline (identification and quantification in separate analytical runs) and provided validation tools together with absolute concentration values for > 350 lipids in human plasma on a species level with an analytical run-time of less than 25 min per sample.

Fatty sweet symphony: Decoding distinct ganglioside patterns of native and differentiated mesenchymal stem cells by a novel glycolipidomics profiling strategy

“…Furthermore, the cells were successfully differentiated towards the chondrogenic, adipogenic, and osteogenic lineage. This study's findings are in accordance with observed ganglioside regulations of our previous study on MSC and adipogenic cell differentiation coming from a different stem cell donor source (female, other adipose tissue part) (Schoeny et al, 2021).…”

Recurrent Topics in Mass Spectrometry-Based Metabolomics and Lipidomics—Standardization, Coverage, and Throughput

“… 89 Here, we want to emphasize the possibility of class or retention-time specific standardization if the target metabolite or lipid is not present in the yeast extract. By using these labeled compounds as class or retention-time specific ISTD if the target analyte is not present in the yeast extract, 90 the list of possible analytes in a quantitative approach can further be enlarged and adapted to the sample of interest.…”

Data Processing of Product Ion Spectra: Quality Improvement by Averaging Multiple Similar Spectra of Small Molecules

“…In metabolomics studies using high-resolution mass spectrometry (MS), a precise mass-tocharge ratio (m/z) value is measured to deduce the compositional formulae of a metabolite-derived ion 1) . In addition, the product ion spectrum of the detected ion is simultaneously acquired using the data-dependent acquisition (DDA) mode of various tandem MS, such as quadrupole-time-of-flight (Q-TOF) MS [2][3][4][5][6][7] . The fragmentation pattern in the product ion spectrum is also used to annotate structural information regarding the metabolite.…”