LipidFinder: a computational workflow for discovery of lipids identifies eicosanoid-phosphoinositides in platelets

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

Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells

Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S–hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type

Exploration of underutilized crop diversity of Capsicum peppers in their primary center of diversity in Bolivia and Peru

The genus Capsicum is a highly diverse complex of domesticated and wild species that displays abundant variation in its main center of domestication and diversity in Bolivia and Peru but that remains under-researched. New collecting expeditions undertaken in 2010 by the Instituto Nacional de Innovación Agraria (INIA) in Peru and the Centro de Investigaciones Fitoecogenéticas de Pairumani (CIFP) in Bolivia have significantly increased the size of the collections. INIA Peru now maintains 712 accessions of the five domesticated species, making it one of the largest and most diverse national collections of native Capsicum pepper varieties in the world. The collection in Bolivia contains 492 accessions, including the five domesticated species, four wild species, and one wild botanical variety of a domesticated species. We report on the identification of promising native Capsicum germplasm for potential use in the development of differentiated products. Identification of promising material representative of native Capsicum diversity in both collections followed several steps: (1) Identification of a core collection of nearly 100 accessions per country representing the different species and their geographic distribution. Dried samples of these accessions were biochemically screened for commercially interesting attributes including capsaicinoid content, polyphenols, antioxidant capacity, carotenoids, lipid content and color; (2) Based on results of the biochemical screening, sub-sets of 44 Bolivian and 39 Peruvian accessions were selected, representing the different species and variation in biochemical attributes; and (3) The selected materials were grown in different environments to identify the agro-ecological conditions were they best express the special properties of commercial interest. The biochemical screening and agromorphological characterization and evaluation revealed that Capsicum accessions from Bolivia and Peru have unique combinations of functional attributes, confirming that a wealth of commercially valuable properties can be found in Capsicum’s primary center of diversity. This study was financed by GIZ

LipidFinder on LIPID MAPS: peak filtering, MS searching and statistical analysis for lipidomics

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

Fatty acids are crucial to fuel NK cells upon acute retrovirus infection

Natural killer (NK) cells are cytotoxic innate immune cells, able to recognize and eliminate virus-infected as well as cancer cells. Metabolic reprogramming is crucial for their activity as they have enhanced energy and nutritional demands for their functions during an infection. Fatty acids (FAs) represent an important source of cellular energy and are essential for proliferation of immune cells. However, the precise role of FAs for NK cells activity in retrovirus infection was unknown. Here we show that activated NK cells increase the expression of the FA uptake receptor CD36 and subsequently the uptake of FAs upon acute virus infection. We found an enhanced flexibility of NK cells to utilize FAs as source of energy compare to naïve NK cells. NK cells that were able to generate energy from FAs showed an augmented target cell killing and increased expression of cytotoxic parameters. However, NK cells that were unable to generate energy from FAs exhibited a severely decreased migratory capacity. Our results demonstrate that NK cells require FAs in order to fight acute virus infection. Susceptibility to severe virus infections as it is shown for people with malnutrition may be augmented by defects in the FA processing machinery, which might be a target to therapeutically boost NK cell functions in the future

Oxylipin metabolism is controlled by mitochondrial β-oxidation during bacterial inflammation

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation

Metabolic dysregulation of the lysophospholipid/autotaxin axis in the chromosome 9p21 gene SNP rs10757274

Background - Common chromosome 9p21 SNPs increase coronary heart disease (CHD) risk, independent of 'traditional lipid risk factors'. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here we applied lipidomic and genomic approaches to three model systems, to characterize lipid metabolic changes in common Chr9p21 SNPs which confer ~30% elevated CHD risk associated with altered expression of ANRIL, a long ncRNA. Methods - Untargeted and targeted lipidomics was applied to plasma from Northwick Park Heart Study II (NPHSII) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from iPSCs of individuals with/without Chr9p21 risk, non-risk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL/lysoPA genes. Results - Elevated risk GG correlated with reduced lysophosphospholipids (lysoPLs), lysophosphatidic acids (lysoPA) and autotaxin (ATX). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolising enzymes was found in HEK cells lacking ANRIL. In the VSMC dataset, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed expression of several lysoPL/lysoPA genes to non-risk haplotype levels. Genes that were altered across both cell datasets were DGKA, MBOAT2, PLPP1 and LPL. The in-silico analysis identified four ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, miR-34a-5p. Conclusions - A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with CHD pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated

Capillary zone electrophoresis coupled to drift tube ion mobility-mass spectrometry for the analysis of native and APTS-labeled N-glycans

Capillary zone electrophoresis (CZE) based on electrophoretic mobility in the liquid phase and ion mobility spectrometry (IMS) based on mobilities in the gas phase are both powerful techniques for the separation of complex samples. Protein glycosylation is one of the most common post-translational modifications associated with a wide range of biological functions and human diseases. Due to their high structural variability, the analysis of glycans still represents a challenging task. In this work, the first on-line coupling of CZE with drift tube ion mobility-mass spectrometry (DTIM-MS) has been perfomed to further improve separation capabilities for the analysis of native and 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled N-glycans. In this way, a complexity of glycan signals was revealed which could not be resolved by these techniques individually, shown for both native and APTS-labeled glycans. Each individual glycan signal separated in CZE exhibited an unexpectedly high number of peaks observed in the IMS dimension. This observation could potentially be explained by the presence of isomeric forms, including different linkages, and/or gas-phase conformers. In addition, the type of sialic acid attached to glycans has a significant impact on the obtained drift time profile. Furthermore, the application of alpha 2-3 neuraminidase enabled the partial assignment of peaks in the arrival time distribution considering their sialic acid linkages (alpha 2-3/alpha 2-6). This work is a showcase for the high potential of CZE-DTIM-MS, which is expected to find various applications in the future

Comparison of derivatization/ionization techniques for liquid chromatography tandem mass spectrometry analysis of oxylipins

The performance of two derivatization and ionization techniques for the quantitative reversed phase liquid chromatography (LC)- mass spectrometry (MS) analysis of hydroxy fatty acids (OH-PUFA) in plasma was evaluated: One used AMPP (N-(4-aminomethylphenyl)pyridinium chloride) leading to a positive charged amid-derivate which can be detected by electrospray ionization (ESI)-MS. Second yielded penta fluorobenzyl bromide (PFB) ester derivates allowing detection in electron capture atmospheric pressure chemical ionization (ecAPCI)-MS. The sensitivity of detection of a comprehensive set of hydroxy fatty acids of n6- and n3- poly unsaturated fatty acids was investigated. On the SCIEX3200 MS the applied PFB derivatization led to poor limits of detection (LOD) of 10–100 nM (0.1–1 pmol/0.03–0.3 ng on column). By contrast, AMPP derivatization led to a similar sensitivity compared to the standard ESI(-) of non derivatized analytes (LOD about 1 nM (10 fmol/3 pg on column)). For several analytes, including 9-HETE, 11-HETE and 17-HDHA the AMPP derivatization improved sensitivity enabling their detection in human plasma. However, precision was reduced by AMPP derivatization and variation in IS recovery indicated a strong matrix influence on the MS-signal. In sum, with the instrumentation used, neither of these derivatization methods improves in our hands the LC–MS based quantification of oxylipins

Comparison of derivatization/ionization techniques for liquid chromatography tandem mass spectrometry analysis of oxylipins

The performance of two derivatization and ionization techniques for the quantitative reversed phase liquid chromatography (LC)- mass spectrometry (MS) analysis of hydroxy fatty acids (OH-PUFA) in plasma was evaluated: One used AMPP (N-(4-aminomethylphenyl)pyridinium chloride) leading to a positive charged amid-derivate which can be detected by electrospray ionization (ESI)-MS. Second yielded penta fluorobenzyl bromide (PFB) ester derivates allowing detection in electron capture atmospheric pressure chemical ionization (ecAPCI)-MS. The sensitivity of detection of a comprehensive set of hydroxy fatty acids of n6- and n3- poly unsaturated fatty acids was investigated. On the SCIEX3200 MS the applied PFB derivatization led to poor limits of detection (LOD) of 10–100 nM (0.1–1 pmol/0.03–0.3 ng on column). By contrast, AMPP derivatization led to a similar sensitivity compared to the standard ESI(-) of non derivatized analytes (LOD about 1 nM (10 fmol/3 pg on column)). For several analytes, including 9-HETE, 11-HETE and 17-HDHA the AMPP derivatization improved sensitivity enabling their detection in human plasma. However, precision was reduced by AMPP derivatization and variation in IS recovery indicated a strong matrix influence on the MS-signal. In sum, with the instrumentation used, neither of these derivatization methods improves in our hands the LC–MS based quantification of oxylipins