Lipids are a large and highly diverse family of biomolecules, which play essential structural, storage and signalling roles in cells and tissues. Although traditional mass spectrometry (MS) approaches used in lipidomics are highly sensitive and selective, lipid analysis remains challenging due to the chemical diversity of lipid structures, multiple isobaric species and incomplete separation using many forms of chromatography. Ion mobility (IM) separates ions in the gas phase based on their physicochemical properties. Addition of IM to the traditional lipidomic workflow both enhances separation of complex lipid mixtures, beneficial for lipid identification, and improves isomer resolution. Herein, we discuss the recent developments in IM-MS for lipidomics.

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A3, DXA3). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA3 formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A2 (cPLA2), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA3 is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.

Analysis of oxylipins by liquid chromatography mass spectrometry (LC-MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC-MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC-DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross section values in nitrogen (DT CCSN2) and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DT CCSN2 and m/z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS, but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium infected murine bone-marrow derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.

IntroductionData processing is one of the biggest problems in metabolomics, given the high number of samples analyzed and the need of multiple software packages for each step of the processing workflow.ObjectivesMerge in the same platform the steps required for metabolomics data processing.MethodsKniMet is a workflow for the processing of mass spectrometry-metabolomics data based on the KNIME Analytics platform.ResultsThe approach includes key steps to follow in metabolomics data processing: feature filtering, missing value imputation, normalization, batch correction and annotation.ConclusionKniMet provides the user with a local, modular and customizable workflow for the processing of both GC–MS and LC–MS open profiling data.

1Background Inflammatory bowel disease is a group of pathologies characterised by chronic 2 inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn's disease 3 and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and 4 metabolomics could represent a less invasive approach to identify biomarkers of disease presence and 5 progression. 6Objectives The lipid and the polar metabolite profile of plasma samples of patients affected by 7 inflammatory bowel disease have been compared with healthy individuals with the aim to find their 8 metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase 9 extraction to further characterise differences between pathological samples. 10 Methods A total of 200 plasma samples were analysed using drift tube ion mobility coupled with 11 time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, 12 while liquid chromatography coupled with triple quadrupole mass spectromety was used for the polar 13 metabolite profile analysis. 14 Results Variations in the lipid profile between inflammatory bowel disease and healthy individuals 15 were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly 16 changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid 17 metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also 18 found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory 19 bowel disease.20Conclusions Given good statistical results and predictive power of the model produced in our study, 21 metabolomics can be considered as a valid tool to investigate inflammatory bowel disease. 22 23 Inflammatory bowel disease (IBD) is a group of pathologies characterised by a chronic phlogosis, 24 and a not specified etiology (Baumgart and Carding 2007). The main clinically defined forms of IBD 25 are Crohn's disease (CD) and ulcerative colitis (UC) (Kaser et al. 2010, Kumar and Clark 2016). The 26 incidence of IBD fluctuates from country to country but the main two typologies affect 1.5 million 27 Americans, 2.2 million Europeans, and several hundred thousands more worldwide (Kumar and 28 Clark 2016, Cosnes et al. 2011). Diagnosis of IBDs is particularly challenging, as other diseases 29 causing similar signs and symptoms need to be excluded first through a combination of tests, but the 30 ultimate diagnostic tool remains endoscopic examination coupled with biopsies. Furthermore, 31 discrimination between the two manifestations, UC and CD, is particularly complicated given the 32 similarity of the symptoms, resulting in 10-15% of cases lacking a defined diagnosis (undefined 33 colitis) (Kumar and Clark 2016). Different causative agents have been proposed in the past for CD 34 and UC diagnosis. One of these theories is based on the T-cell pathway, that proposes the idea that T 35 cells activation...

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.

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (ob/ob), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.