Tandem mass spectral library search (MS/MS) is the fastest way to correctly annotate MS/MS spectra from screening small molecules in fields such as environmental analysis, drug screening, lipid ...analysis, and metabolomics. The confidence in MS/MS‐based annotation of chemical structures is impacted by instrumental settings and requirements, data acquisition modes including data‐dependent and data‐independent methods, library scoring algorithms, as well as post‐curation steps. We critically discuss parameters that influence search results, such as mass accuracy, precursor ion isolation width, intensity thresholds, centroiding algorithms, and acquisition speed. A range of publicly and commercially available MS/MS databases such as NIST, MassBank, MoNA, LipidBlast, Wiley MSforID, and METLIN are surveyed. In addition, software tools including NIST MS Search, MS‐DIAL, Mass Frontier, SmileMS, Mass++, and XCMS2 to perform fast MS/MS search are discussed. MS/MS scoring algorithms and challenges during compound annotation are reviewed. Advanced methods such as the in silico generation of tandem mass spectra using quantum chemistry and machine learning methods are covered. Community efforts for curation and sharing of tandem mass spectra that will allow for faster distribution of scientific discoveries are discussed.
The mammalian brain relies on neurochemistry to fulfill its functions. Yet, the complexity of the brain metabolome and its changes during diseases or aging remain poorly understood. Here, we generate ...a metabolome atlas of the aging wildtype mouse brain from 10 anatomical regions spanning from adolescence to old age. We combine data from three assays and structurally annotate 1,547 metabolites. Almost all metabolites significantly differ between brain regions or age groups, but not by sex. A shift in sphingolipid patterns during aging related to myelin remodeling is accompanied by large changes in other metabolic pathways. Functionally related brain regions (brain stem, cerebrum and cerebellum) are also metabolically similar. In cerebrum, metabolic correlations markedly weaken between adolescence and adulthood, whereas at old age, cross-region correlation patterns reflect decreased brain segregation. We show that metabolic changes can be mapped to existing gene and protein brain atlases. The brain metabolome atlas is publicly available ( https://mouse.atlas.metabolomics.us/ ) and serves as a foundation dataset for future metabolomic studies.
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•Epimetabolites are modified classic metabolites with new roles.•Example roles include regulation of tumorigenesis, inflammation and pluripotency.•New epimetabolites are discovered by ...untargeted metabolomics.•Structure annotation by in-silico prediction software.
Enzymatic transformations of primary, canonical metabolites generate active biomolecules that regulate important cellular and physiological processes. Roles include regulation of histone demethylation in epigenetics, inflammation in tissue injury, insulin sensitivity, cancer cell invasion, stem cell pluripotency status, inhibition of nitric oxide signaling and others. Such modified compounds, defined as epimetabolites, have functions distinct from classic hormones as well as removed from generic anabolism and catabolism. Epimetabolites are discovered by untargeted metabolomics using liquid- or gas chromatography–high resolution mass spectrometry and structurally annotated by in-silico fragmentation prediction tools. Their specific biological functions are subsequently investigated by targeted metabolomics methods.
Obesity and accompanying metabolic disease is negatively correlated with lung health yet the exact mechanisms by which obesity affects the lung are not well characterized. Since obesity is associated ...with lung diseases as chronic bronchitis and asthma, we designed a series of experiments to measure changes in lung metabolism in mice fed obesogenic diets. Mice were fed either control or high fat/sugar diet (45%kcal fat/17%kcal sucrose), or very high fat diet (60%kcal fat/7% sucrose) for 150 days. We performed untargeted metabolomics by GC-TOFMS and HILIC-QTOFMS and lipidomics by RPLC-QTOFMS to reveal global changes in lung metabolism resulting from obesity and diet composition. From a total of 447 detected metabolites, we found 91 metabolite and lipid species significantly altered in mouse lung tissues upon dietary treatments. Significantly altered metabolites included complex lipids, free fatty acids, energy metabolites, amino acids and adenosine and NAD pathway members. While some metabolites were altered in both obese groups compared to control, others were different between obesogenic diet groups. Furthermore, a comparison of changes between lung, kidney and liver tissues indicated few metabolic changes were shared across organs, suggesting the lung is an independent metabolic organ. These results indicate obesity and diet composition have direct mechanistic effects on composition of the lung metabolome, which may contribute to disease progression by lung-specific pathways.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Urine metabolites are used in many clinical and biomedical studies but usually only for a few classic compounds. Metabolomics detects vastly more metabolic signals that may be used to precisely ...define the health status of individuals. However, many compounds remain unidentified, hampering biochemical conclusions. Here, we annotate all metabolites detected by two untargeted metabolomic assays, hydrophilic interaction chromatography (HILIC)-Q Exactive HF mass spectrometry and charged surface hybrid (CSH)-Q Exactive HF mass spectrometry. Over 9,000 unique metabolite signals were detected, of which 42% triggered MS/MS fragmentations in data-dependent mode. On the highest Metabolomics Standards Initiative (MSI) confidence level 1, we identified 175 compounds using authentic standards with precursor mass, retention time, and MS/MS matching. An additional 578 compounds were annotated by precursor accurate mass and MS/MS matching alone, MSI level 2, including a novel library specifically geared at acylcarnitines (CarniBlast). The rest of the metabolome is usually left unannotated. To fill this gap, we used the in silico fragmentation tool CSI:FingerID and the new NIST hybrid search to annotate all further compounds (MSI level 3). Testing the top-ranked metabolites in CSI:Finger ID annotations yielded 40% accuracy when applied to the MSI level 1 identified compounds. We classified all MSI level 3 annotations by the NIST hybrid search using the ClassyFire ontology into 21 superclasses that were further distinguished into 184 chemical classes. ClassyFire annotations showed that the previously unannotated urine metabolome consists of 28% derivatives of organic acids, 16% heterocyclics, and 16% lipids as major classes.
Although understudied relative to many phospholipids, accumulating evidence suggests that bis(monoacylglycero)phosphate (BMP) is an important class of regulatory lipid that plays key roles in ...lysosomal integrity and function. BMPs are rare in most mammalian tissues, comprising only a few percent of total cellular lipid content, but are elevated in cell types such as macrophages that rely heavily on lysosomal function. BMPs are markedly enriched in endosomal and lysosomal vesicles compared to other organelles and membranous structures, and their unique
-1:
-1' stereoconfiguration may confer stability within the hydrolytic lysosomal environment. BMP-enriched vesicles serve in endosomal-lysosomal trafficking and function as docking structures for the activation of lysosomal hydrolytic enzymes, notably those involved in the catabolic breakdown of sphingolipids. BMP levels are dysregulated in lysosomal storage disorders, phospholipidosis, metabolic diseases, liver and kidney diseases and neurodegenerative disorders. However, whether BMP alteration is a mediator or simply a marker of pathological states is unclear. Likewise, although BMP acyl chain composition may be altered with disease states, the functional significance of specific BMP species remains to be resolved. Newly developed tools for untargeted lipidomic analysis, together with a deeper understanding of enzymes mediating BMP synthesis and degradation, will help shed further light on the functional significance of BMPs in cellular physiology and pathology.
Inborn errors of metabolism (IEMs) are a group of inherited diseases with variable incidences. IEMs are caused by disrupting enzyme activities in specific metabolic pathways by genetic mutations, ...either directly or indirectly by cofactor deficiencies, causing altered levels of compounds associated with these pathways. While IEMs may present with multiple overlapping symptoms and metabolites, early and accurate diagnosis of IEMs is critical for the long-term health of affected subjects. The prevalence of IEMs differs between countries, likely because different IEM classifications and IEM screening methods are used. Currently, newborn screening programs exclusively use targeted metabolic assays that focus on limited panels of compounds for selected IEM diseases. Such targeted approaches face the problem of false negative and false positive diagnoses that could be overcome if metabolic screening adopted analyses of a broader range of analytes. Hence, we here review the prospects of using untargeted metabolomics for IEM screening. Untargeted metabolomics and lipidomics do not rely on predefined target lists and can detect as many metabolites as possible in a sample, allowing to screen for many metabolic pathways simultaneously. Examples are given for nontargeted analyses of IEMs, and prospects and limitations of different metabolomics methods are discussed. We conclude that dedicated studies are needed to compare accuracy and robustness of targeted and untargeted methods with respect to widening the scope of IEM diagnostics.
Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated ...stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX. Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.
Nonalcoholic fatty liver disease (NAFLD) in non-obese patients remains a clinical condition with unclear etiology and pathogenesis. Using a metabolomics approach in a mouse model that recapitulates ...almost all the characteristic features of non-obese NAFLD, we aimed to advance mechanistic understanding of this disorder. Mice fed high fat, high cholesterol, cholate (HFHCC) diet for three weeks consistently developed hepatic pathology similar to NAFLD and nonalcoholic steatohepatitis (NASH) without changes to body weight or fat pad weights. Gas- and liquid chromatography/mass spectrometry-based profiling of lipidomic and primary metabolism changes in the liver and plasma revealed that systemic mechanisms leading to steatosis and hepatitis in this non-obese NAFLD model were driven by a combination of effects directed by elevated free cholesterol, cholesterol esters and cholic acid, and associated changes to metabolism of sphingomyelins and phosphatidylcholines. These results demonstrate that mechanisms underlying cholesterol-induced non-obese NAFLD are distinct from NAFLD occurring as a consequence of metabolic syndrome. In addition, this investigation provides one of the first metabolite reference profiles for interpreting effects of dietary and hepatic cholesterol in human non-obese NAFLD/NASH patients.
Regulation of embryonic diapause, dormancy that interrupts the tight connection between developmental stage and time, is still poorly understood. Here, we characterize the transcriptional and ...metabolite profiles of mouse diapause embryos and identify unique gene expression and metabolic signatures with activated lipolysis, glycolysis, and metabolic pathways regulated by AMPK. Lipolysis is increased due to mTORC2 repression, increasing fatty acids to support cell survival. We further show that starvation in pre-implantation ICM-derived mouse ESCs induces a reversible dormant state, transcriptionally mimicking the in vivo diapause stage. During starvation, Lkb1, an upstream kinase of AMPK, represses mTOR, which induces a reversible glycolytic and epigenetically H4K16Ac-negative, diapause-like state. Diapause furthermore activates expression of glutamine transporters SLC38A1/2. We show by genetic and small molecule inhibitors that glutamine transporters are essential for the H4K16Ac-negative, diapause state. These data suggest that mTORC1/2 inhibition, regulated by amino acid levels, is causal for diapause metabolism and epigenetic state.
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•Diapause is associated with increased lipolysis and glutamine transporter expression•Upregulation of lipolysis in diapause is associated with downregulation of mTORC2•Starvation results in a diapause-like state, enriched in glutamine transporters•Inhibition of glutamine transporters leads to exit from the diapause epigenetic state
Hussein et al. report that, during starvation, mTOR is repressed through LKB1-AMPK, inducing a reversible metabolically active but epigenetically silenced embryonic diapause-like state that upregulates expression of the glutamine transporters SLC38A1/2. These transporters are required for the H4K16ac-negative, diapause state.
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