SUMMARY:We present LipidFinder online, hosted on the LIPID MAPS website, as a liquid chromatography/mass spectrometry (LC/MS) workflow comprising peak filtering, MS searching and statistical analysis components, highly customized for interrogating lipidomic data. The online interface of LipidFinder includes several innovations such as comprehensive parameter tuning, a MS search engine employing in-house customized, curated and computationally generated databases and multiple reporting/display options. A set of integrated statistical analysis tools which enable users to identify those features which are significantly-altered under the selected experimental conditions, thereby greatly reducing the complexity of the peaklist prior to MS searching is included. LipidFinder is presented as a highly flexible, extensible user-friendly online workflow which leverages the lipidomics knowledge base and resources of the LIPID MAPS website, long recognized as a leading global lipidomics portal.AVAILABILITY AND IMPLEMENTATION:LipidFinder on LIPID MAPS is available at: http://www.lipidmaps.org/data/LF.

Background Phospholipids on the surface of platelets play a key role in supporting coagulation in both health and disease. Alterations to phospholipid composition in platelets can contribute to thrombotic and bleeding disorders. We recently identified pro-coagulant enzymatically oxidized phospholipids (eoxPL) generated by platelets in response to thrombin activation via cyclooxygenase-1 (COX) and 12-lipoxygenase (LOX). It is not known how these lipids vary in relation to aspirin (a COX inhibitor) supplementation and/or gender. Methods Twenty-eight healthy volunteers (14 males, 14 females) donated blood for platelet isolation at baseline (following a 14-day NSAID washout) and after 7-day supplementation with 75 mg aspirin. Repeat sampling took place 2 months and 4 months later to account for variations over time. Lipids were extracted from platelets either basally or following 30 min thrombin activation then analysed using liquid chromatography with tandem mass-spectrometry (LC-MS/MS) for the 49 most abundant eoxPL molecular species. Results The majority (96%) of eoxPL increased in response to thrombin activation of platelets. Gender differences were observed with a trend towards higher levels generated by females. Correlation analysis highlighted clustering of eoxPL by enzymatic origin (12-LOX versus COX-1). Aspirin supplementation decreased eoxPL generated via COX-1 in both genders, but in males it increased levels of those generated by 12-LOX (p<0.05). Importantly, elevated levels of 12-hydroxyeicosatetraenoic acid (12-HETE)-containing eoxPL, which are known to be procoagulant, were observed. In contrast, generation of free 12-HETE was not affected by aspirin. Conclusion In this study, we found an influence of both gender and aspirin supplementation on the generation of eoxPL in thrombin-activated platelets from healthy volunteers. Further characterisation of the gender influence may reveal novel mechanisms that contribute to the risk of arterial thrombosis in human populations. Conflict of interest None declared

Accurate and high-quality curation of lipidomic datasets generated from plasma, cells, or tissues is becoming essential for cell biology investigations and biomarker discovery for personalized medicine. However, a major challenge lies in removing artifacts otherwise mistakenly interpreted as real lipids from large mass spectrometry files (> 60 K features), while retaining genuine ions in the dataset. This requires powerful informatics tools; however, available workflows have not been tailored specifically for lipidomics, particularly discovery research. We designed LipidFinder, an open-source Python workflow. An algorithm is included that optimizes analysis based on users ' own data, and outputs are screened against online databases and categorized into LIPID MAPS classes. LipidFinder outperformed three widely used metabolomics packages using data from human platelets. We show a family of three 12-hydroxyeicosatetraenoic acid phosphoinositides (16:0/, 18:1/, 18:0/12-HETE-PI) generated by thrombin-activated platelets, indicating crosstalk between eicosanoid and phosphoinositide pathways in human cells. The software is available on GitHub (https://github.com/cjbrasher/LipidFinder), with full userguides.

Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via β-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented.