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Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.
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Biología Computacional , Lipidómica , Biología Computacional/métodos , Programas Informáticos , Informática , Lípidos/químicaRESUMEN
TopBP1 has important roles in both DNA replication and checkpoint regulation in vertebrates. We have identified a protein called Treslin that associates with TopBP1 in Xenopus egg extracts. Depletion of Treslin from egg extracts strongly inhibits chromosomal DNA replication. Binding of Treslin to chromatin in egg extracts occurs independently of TopBP1. However, loading of the initiator protein Cdc45 onto chromatin cannot take place in the absence of Treslin. Prior to the initiation of DNA replication, Treslin associates with TopBP1 in a Cdk2-dependent manner. Ablation of Treslin from human cells also strongly inhibits DNA replication. Taken together, these results indicate that Treslin and TopBP1 collaborate in the Cdk2-mediated loading of Cdc45 onto replication origins. Thus, Treslin regulates a pivotal step in the initiation of DNA replication in vertebrates.
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Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , Datos de Secuencia Molecular , Origen de Réplica , Fase S , XenopusRESUMEN
The rapid increase in lipidomic studies has led to a collaborative effort within the community to establish standards and criteria for producing, documenting, and disseminating data. Creating a dynamic easy-to-use checklist that condenses key information about lipidomic experiments into common terminology will enhance the field's consistency, comparability, and repeatability. Here, we describe the structure and rationale of the established Lipidomics Minimal Reporting Checklist to increase transparency in lipidomics research.
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We present an instrument-independent benchmark procedure and software (LFQ_bout) for the validation and comparative evaluation of the performance of LC-MS/MS and data processing workflows in bottom-up proteomics. The procedure enables a back-to-back comparison of common and emerging workflows, e.g., diaPASEF or ScanningSWATH, and evaluates the impact of arbitrary and inadequately documented settings or black-box data processing algorithms. It enhances the overall performance and quantification accuracy by recognizing and reporting common quantification errors.
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Cromatografía Líquida con Espectrometría de Masas , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Proteoma , Proteómica/métodos , Benchmarking , Programas InformáticosRESUMEN
Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster, which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food. We developed a series of semisynthetic foods to manipulate the length and unsaturation of fatty acid moieties in GPLs and singled out proteins whose abundance is specifically affected by membrane lipid unsaturation in the Drosophila eye. Unexpectedly, we identified a group of proteins that have muscle-related functions and increased their abundances under unsaturated eye lipidome conditions. In contrast, the abundance of two stress response proteins, Turandot A and Smg5, is decreased by lipid unsaturation. Our findings could guide the genetic dissection of homeostatic mechanisms that maintain visual function when the eye is exposed to environmental and dietary challenges.
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Drosophila , Proteoma , Animales , Proteoma/genética , Drosophila melanogaster/genética , Lipidómica , Ácidos Grasos , GlicerofosfolípidosRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.
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Metabolismo de los Lípidos/genética , Neuronas/metabolismo , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Animales , Animales Modificados Genéticamente/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Gotas Lipídicas/metabolismo , Lipólisis/genética , Proteínas de Transporte de Membrana/genética , Neuroblastoma/genética , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Perilipina-2/genética , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , ProteolisisRESUMEN
Drosophila melanogaster is a popular model organism to elucidate the molecular mechanisms that underlie the structure and function of the eye as well as the causes of retinopathies, aging, light-induced damage, or dietary deficiencies. Large-scale screens have isolated genes whose mutation causes morphological and functional ocular defects, which led to the discovery of key components of the phototransduction cascade. However, the proteome of the Drosophila eye is poorly characterized. Here, we used GeLC-MS/MS to quantify 3516 proteins, including the absolute (molar) quantities of 43 proteins in the eye of adult male Drosophila reared on standard laboratory food. This work provides a generic and expandable resource for further genetic, pharmacological, and dietary studies.
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Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.
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Proteínas , Segmento Externo de la Célula en Bastón , Animales , Ratones , Proteínas/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Fototransducción , Retina/metabolismoRESUMEN
Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.
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Diatomeas , Biomineralización , Cromatografía Liquida , Diatomeas/metabolismo , Proteoma/metabolismo , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Espectrometría de Masas en TándemRESUMEN
Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Ciclo Celular , Cromosomas/ultraestructura , Replicación del ADN , Eliminación de Gen , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiaeRESUMEN
Treslin helps to trigger the initiation of DNA replication by promoting integration of Cdc45 into the replicative helicase. Treslin is a key positive-regulatory target of cell-cycle control mechanisms; activation of Treslin by cyclin-dependent kinase is essential for the initiation of replication. Here we demonstrate that Treslin is also a critical locus for negative regulatory mechanisms that suppress initiation. We found that the checkpoint-regulatory kinase Chk1 associates specifically with a C-terminal domain of Treslin (designated TRCT). Mutations in the TRCT domain abolish binding of Chk1 to Treslin and thereby eliminate Chk1-catalyzed phosphorylation of Treslin. Significantly, abolition of the Treslin-Chk1 interaction results in elevated initiation of chromosomal DNA replication during an unperturbed cell cycle, which reveals a function for Chk1 during a normal S phase. This increase is due to enhanced loading of Cdc45 onto potential replication origins. These studies provide important insights into how vertebrate cells orchestrate proper initiation of replication.
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Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas Quinasas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromosomas/metabolismo , Células HEK293 , Humanos , Fosforilación , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.
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Diglicéridos/química , Lípidos/química , Proteínas/metabolismo , Adenosina Trifosfato/metabolismo , Técnicas Biosensibles , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Supervivencia Celular , Isoenzimas/metabolismo , Cinética , Luz , Modelos Biológicos , Proteína Quinasa C/metabolismo , Transducción de SeñalRESUMEN
By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.
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Péptidos , Proteoma , Calibración , Marcaje Isotópico/métodos , Péptidos/química , Estándares de ReferenciaRESUMEN
Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. The FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa), stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q)-peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R)-peptides that relate its abundance to a single protein standard (bovine serum albumin, BSA). FastCAT not only alleviates the need to purify CP or use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but also improves the accuracy, precision, and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of the target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in human cerebrospinal fluid at the low ng/mL level.
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Proteínas , Proteómica , Electroforesis en Gel de Poliacrilamida , Humanos , Péptidos/metabolismo , Proteómica/métodos , Estándares de ReferenciaRESUMEN
OBJECTIVE: Lipidomic changes were causally linked to metabolic diseases, but the scenario for colorectal cancer (CRC) is less clear. We investigated the CRC lipidome for putative tumor-specific alterations through analysis of 3 independent retrospective patient cohorts from 2 clinical centers, to derive a clinically useful signature. DESIGN: Quantitative comprehensive lipidomic analysis was performed using direct infusion electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS) and high-resolution mass spectrometry (HR-MS) on matched nondiseased mucosa and tumor tissue in a discovery cohort (n = 106). Results were validated in 2 independent cohorts (n = 28, and n = 20), associated with genomic and clinical data, and lipidomic data from a genetic mouse tumor model (Apc1638N). RESULTS: Significant differences were found between tumor and normal tissue for glycero-, glycerophospho-, and sphingolipids in the discovery cohort. Comparison to the validation collectives unveiled that glycerophospholipids showed high interpatient variation and were strongly affected by preanalytical conditions, whereas glycero- and sphingolipids appeared more robust. Signatures of sphingomyelin and triacylglycerol (TG) species significantly differentiated cancerous from nondiseased tissue in both validation studies. Moreover, lipogenic enzymes were significantly up-regulated in CRC, and FASN gene expression was prognostically detrimental. The TG profile was significantly associated with postoperative disease-free survival and lymphovascular invasion, and was essentially conserved in murine digestive cancer, but not associated with microsatellite status, KRAS or BRAF mutations, or T-cell infiltration. CONCLUSION: Analysis of the CRC lipidome revealed a robust TG-species signature with prognostic potential. A better understanding of the cancer-associated glycerolipid and sphingolipid metabolism may lead to novel therapeutic strategies.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Lipidómica , Lípidos/análisis , Metaboloma , Adulto , Anciano , Anciano de 80 o más Años , Animales , Ceramidas/análisis , Colectomía , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Supervivencia sin Enfermedad , Femenino , Genes APC , Alemania , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Invasividad Neoplásica , Reproducibilidad de los Resultados , Estudios Retrospectivos , Espectrometría de Masa por Ionización de Electrospray , Esfingolípidos/análisis , Espectrometría de Masas en Tándem , Triglicéridos/análisisRESUMEN
Although lipids are biomolecules with seemingly simple chemical structures, the molecular composition of the cellular lipidome is complex and, currently, poorly understood. The exact mechanisms of how compositional complexity affects cell homeostasis and its regulation also remain unclear. This emerging field is developing sensitive mass spectrometry technologies for the quantitative characterization of the lipidome. Here, we argue that lipidomics will become an essential tool kit in cell and developmental biology, molecular medicine and nutrition.
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Metabolismo de los Lípidos , Lípidos/química , Enfermedad/etiología , Humanos , Lípidos/análisis , Espectrometría de Masas , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Metabolómica , Estructura MolecularRESUMEN
Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.
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Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Estrés del Retículo Endoplásmico/inmunología , Activación de Linfocitos , Células T Asesinas Naturales/inmunología , Animales , Presentación de Antígeno , Antígenos CD1d/biosíntesis , Antígenos CD1d/inmunología , Autoantígenos/inmunología , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Citoesqueleto/ultraestructura , Endosomas/inmunología , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Lípidos/inmunología , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Células THP-1 , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/inmunología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/fisiologíaRESUMEN
In Drosophila larvae, growth and developmental timing are regulated by nutrition in a tightly coordinated fashion. The networks that couple these processes are far from understood. Here, we show that the intestine responds to nutrient availability by regulating production of a circulating lipoprotein-associated form of the signaling protein Hedgehog (Hh). Levels of circulating Hh tune the rates of growth and developmental timing in a coordinated fashion. Circulating Hh signals to the fat body to control larval growth. It regulates developmental timing by controlling ecdysteroid production in the prothoracic gland. Circulating Hh is especially important during starvation, when it is also required for mobilization of fat body triacylglycerol (TAG) stores. Thus, we demonstrate that Hh, previously known only for its local morphogenetic functions, also acts as a lipoprotein-associated endocrine hormone, coordinating the response of multiple tissues to nutrient availability.
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Drosophila melanogaster/crecimiento & desarrollo , Proteínas Hedgehog/sangre , Fenómenos Fisiológicos de la Nutrición/fisiología , Animales , Drosophila melanogaster/metabolismo , Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , LarvaRESUMEN
OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.
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Aciltransferasas/genética , Cirrosis Hepática/genética , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Aciltransferasas/deficiencia , Adulto , Anciano , Animales , Biopsia , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Genotipo , Haplotipos , Humanos , Inflamación/genética , Masculino , Proteínas de la Membrana/deficiencia , Ratones Endogámicos C57BL , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a common metabolic dysfunction leading to hepatic steatosis. However, NAFLD's global impact on the liver lipidome is poorly understood. Using high-resolution shotgun mass spectrometry, we quantified the molar abundance of 316 species from 22 major lipid classes in liver biopsies of 365 patients, including nonsteatotic patients with normal or excessive weight, patients diagnosed with NAFL (nonalcoholic fatty liver) or NASH (nonalcoholic steatohepatitis), and patients bearing common mutations of NAFLD-related protein factors. We confirmed the progressive accumulation of di- and triacylglycerols and cholesteryl esters in the liver of NAFL and NASH patients, while the bulk composition of glycerophospho- and sphingolipids remained unchanged. Further stratification by biclustering analysis identified sphingomyelin species comprising n24:2 fatty acid moieties as membrane lipid markers of NAFLD. Normalized relative abundance of sphingomyelins SM 43:3;2 and SM 43:1;2 containing n24:2 and n24:0 fatty acid moieties, respectively, showed opposite trends during NAFLD progression and distinguished NAFL and NASH lipidomes from the lipidome of nonsteatotic livers. Together with several glycerophospholipids containing a C22:6 fatty acid moiety, these lipids serve as markers of early and advanced stages of NAFL.