Published papers

Mass spectrometry raw data repositories, including Metabolomics Workbench and MetaboLights, have contributed to increased transparency in metabolomics studies and the discovery of novel insights in biology by reanalysis with updated computational metabolomics tools. Herein, we reanalyzed the previously published lipidomics data from nine algal species, resulting in the annotation of 1437 lipids achieving a 40% increase in annotation compared to the previous results. Specifically, diacylglyceryl-carboxyhydroxy-methylcholine (DGCC) in Pavlova lutheri and Pleurochrysis carterae, glucuronosyldiacylglycerol (GlcADG) in Euglena gracilis, and P. carterae, phosphatidylmethanol (PMeOH) in E. gracilis, and several oxidized phospholipids (oxidized phosphatidylcholine, OxPC; phosphatidylethanolamine, OxPE; phosphatidylglycerol, OxPG; phosphatidylinositol, OxPI) in Chlorella variabilis were newly characterized with the enriched lipid spectral databases. Moreover, we integrated the data from untargeted and targeted analyses from data independent tandem mass spectrometry (DIA-MS/MS) acquisition, specifically the sequential window acquisition of all theoretical fragment-ion MS/MS (SWATH-MS/MS) spectra, to increase the lipidomic annotation coverage. After the creation of a global library of precursor and diagnostic ions of lipids by the MS-DIAL untargeted analysis, the co-eluted DIA-MS/MS spectra were resolved in MRMPROBS targeted analysis by tracing the specific product ions involved in acyl chain compositions. Our results indicated that the metabolite quantifications based on DIA-MS/MS chromatograms were somewhat inferior to the MS1-centric quantifications, while the annotation coverage outperformed those of the untargeted analysis of the data dependent and DIA-MS/MS data. Consequently, integrated analyses of untargeted and targeted approaches are necessary to extract the maximum amount of metabolome information, and our results showcase the value of data repositories for the discovery of novel insights in lipid biology.

Mass spectrometry-based untargeted lipidomics has revealed the lipidome atlas of living organisms at the molecular species level. Despite the double bond (C = C) position being a crucial factor in biological system, the C = C defined structures have not yet been characterized comprehensively. Here, we present an approach for C = C position-resolved untargeted lipidomics using a combination of oxygen attachment dissociation and computational mass spectrometry to increase the annotation rate. We validated the accuracy of our platform as per the authentic standards of 85 lipids and the biogenic standards of 52 molecules containing polyunsaturated fatty acids (PUFAs) from the cultured cells fed with various fatty acid-enriched media. By analyzing human and mice-derived samples, we characterized 648 unique lipids with the C = C position-resolved level encompassing 24 lipid subclasses defined by LIPIDMAPS. Our platform also illuminated the unique profiles of tissue-specific lipids containing n-3 and/or n-6 very long-chain PUFAs (carbon ≥ 28 and double bonds ≥ 4) in the eye, testis, and brain of the mouse.

Docosahexaenoic acid (DHA) and ultra-long-chain polyunsaturated fatty acids (ULC–PUFAs) are uniquely enriched in membrane phospholipids of retinal photoreceptors. Several studies have shown that di-DHA- and ULC–PUFA-containing phospholipids in photoreceptors have an important role in maintaining normal visual function; however, the molecular mechanisms underlying the synthesis and enrichment of these unique lipids in the retina, and their specific roles in retinal function remain unclear. Long-chain acyl-coenzyme A (CoA) synthetase 6 (ACSL6) preferentially converts DHA into DHA–CoA, which is a substrate during DHA-containing lipid biosynthesis. Here, we report that Acsl6 mRNA is expressed in the inner segment of photoreceptor cells and the retinal pigment epithelial cells, and genetic deletion of ACSL6 resulted in the selective depletion of di-DHA– and ULC–PUFA-containing phospholipids, but not mono-DHA-containing phospholipids in the retina. MALDI mass spectrometry imaging (MALDI–MSI) revealed the selective distribution of di-DHA– and ULC–PUFA-containing phospholipids in the photoreceptor outer segment (OS). Electroretinogram of Acsl6−/− mice exhibited photoreceptor cell-derived visual impairment, whereas the expression levels and localization of opsin proteins were unchanged. Acsl6−/− mice exhibited an age-dependent progressive decrease of the thickness of the outer nuclear layers, whereas the inner nuclear layers and OSs were normal. These results demonstrate that ACSL6 facilitates the local enrichment of di-DHA– and ULC–PUFA-containing phospholipids in the retina, which supports normal visual function and retinal homeostasis.

Lipidomics and metabolomics communities comprise various informatics tools; however, software programs handling multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 for in-depth lipidome structural elucidation through electron-activated dissociation (EAD)-based tandem MS and determining their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section values. With the optimized EAD settings using 14 eV kinetic energy, the program correctly delineated lipid structures for 96.4% of authentic standards, among which 78.0% had the sn-, OH-, and/or C = C positions correctly assigned at concentrations exceeding 1 μM. We showcased our workflow by annotating the sn- and double-bond positions of eye-specific phosphatidylcholines containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and n-3-VLC-PUFA-supplemented HeLa cells, we identified glycerol 3-phosphate acyltransferase as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into the sn1 position of phospholipids in mammalian cells, which was confirmed using EAD-MS/MS and recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering insights into lipid biology.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a key technology in spatial lipidomics that provides high sensitivity and spatial resolution. Because of the ionization preference of lipid classes and the lack of detailed structural information without MS2 data, dual-polarity analyses and the acquisition of MS/MS spectra are essential to improve coverage and annotation accuracy. Multitime MSI analyses of the same tissue section can provide seamless integration of multimodal spatial data. However, the feasibility of performing serial MALDI-MSI for the same pixels with high spatial resolution has not been fully evaluated. Here, we present SMASH imaging for dual-polarity and MS2 spatial lipidomics in a single tissue section. We estimated two characteristic matrix compounds, 2,5-dihydroxyacetophenone and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile, for dual-polarity analyses and defined the feasible number of layers of SMASH imaging via the coefficient of determination (R2). SMASH imaging successfully visualized over 400 lipid species on average with a 30 μm resolution, with annotation criteria S/N ≥ 50, m/z ≤ 10 mDa, and collision cross section ≤ 20 Å2. In four layers of SMASH imaging, the molecular species of 18 lipids were characterized based on MS2 spectra evidence using parallel accumulation-serial fragmentation on a single mouse brain section. Moreover, eight layers of SMASH imaging with a 5 μm spatial resolution annotated 25 lipid species, supported by a spatial correlation metric between multimodal data. Our approach provides multimodal spatial lipidomics to create a lipidome atlas with accurate annotation and high spatial resolution on a single tissue section.

The ocular surface is in direct contact with the external environment and is susceptible to injury from dust, dryness, or other foreign objects. Once corneal injury occurs, a local inflammatory response is triggered, followed by effective repair of the epithelial layer. In this study, we demonstrated that antibiotic treatment delayed corneal wound healing in mice. LC–MS/MS-based untargeted lipidomics and qPCR analyses revealed that the levels of cholesterol sulfate (CS) and the CS-synthesizing enzyme SULT2B1 were significantly upregulated by antibiotic treatment, and SULT2B1 knockout mice exhibited accelerated corneal wound healing along with increased recruitment of neutrophils and eosinophils. Topical application of CS delayed corneal wound healing. In vitro scratch assays revealed that CS delayed the wound healing of human corneal epithelial cells, potentially by inhibiting the DOCK2-Rac pathway. These results highlight the role of commensal bacteria in controlling corneal wound healing via the cholesterol-sulfotransferase pathway.

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2−/− and GPSN2+/− mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2−/− embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/− neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/− neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.