A comparative analysis of different lipidomics workflow for optimized identification and quantitation

Lipidomics analysis involves processing lipid species from complex biological matrices. There are several approaches available to perform lipidomics analysis. Mass-spectrometry based lipidomics analysis is one of the powerful tools to perform both untargeted and targeted lipidomics. Although high-resolution mass spectrometry is well-suited for the robust profiling and relative quantification of the lipidome, the success of lipidomics analysis relies heavily on post-acquisition factors, particularly the data processing tools and workflows. There are several algorithms and software are developed to analyze lipid species. However, the optimized workflow is still underway. In this study, we have compared three different lipidomics workflows to evaluate the accurate identification and quantitation of lipids from complex NIST plasma. Lipids from NIST plasma sample were extracted using acidified CHCl3/MeOH method with addition of Equisplash (Avanti) internal standard. Extracted lipids were separated by RP-UHPLC and analyzed in both ESI+/ESI-mode using a high-resolution Exploris480 MS (Thermo) operated in DDA mode. Raw data were processed using MS-Dial v5.5, Compound Discoverer v3.5 and LipidSearch 5.1. Data analysis was performed using in house R script. Our preliminary results exhibited 31,140 features from more than 80% of the samples with RSD less than 30%. The curated datasets, include only MS/MS-annotated lipids, were used for further comparison. Comparative analysis of untargeted data revealed that over ~93% data shared similarity across all three different platforms. The most common lipid classes are PC, PE, PI, TG, DG, and CE. While lipidomics software showed similar class coverage, performance varied slightly when identifying isobaric lipids across different platforms. Analysis of the curated datasets yielded 1,670 (Compound Discoverer), 1,663 (MS-DIAL), and 1,522 (LipidSearch) lipid species. Although MS-Dial is a free, comprehensive tool for broad lipidomic summaries, paid platforms such as LipidSearch and Compound Discoverer provides deeper, specialized insights into specific lipids such as acyl-CoAs and carnitines.