PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

Identification of bioactive metabolites using activity metabolomics.

The metabolome, the collection of small-molecule chemical entities involved in metabolism, has traditionally been studied with the aim of identifying biomarkers in the diagnosis and prediction of disease. However, the value of metabolome analysis (metabolomics) has been redefined from a simple biomarker identification tool to a technology for the discovery of active drivers of biological processes. It is now clear that the metabolome affects cellular physiology through modulation of other 'omics' levels, including the genome, epigenome, transcriptome and proteome. In this Review, we focus on recent progress in using metabolomics to understand how the metabolome influences other omics and, by extension, to reveal the active role of metabolites in physiology and disease. This concept of utilizing metabolomics to perform activity screens to identify biologically active metabolites - which we term activity metabolomics - is already having a broad impact on biology.

α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming.

Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming.

Metabolomics: beyond biomarkers and towards mechanisms.

Metabolomics, which is the profiling of metabolites in biofluids, cells and tissues, is routinely applied as a tool for biomarker discovery. Owing to innovative developments in informatics and analytical technologies, and the integration of orthogonal biological approaches, it is now possible to expand metabolomic analyses to understand the systems-level effects of metabolites. Moreover, because of the inherent sensitivity of metabolomics, subtle alterations in biological pathways can be detected to provide insight into the mechanisms that underlie various physiological conditions and aberrant processes, including diseases.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

A global perspective on the status of clinical metabolomics in laboratory medicine - a survey by the IFCC metabolomics working group.

OBJECTIVES: Metabolomics aims for comprehensive characterization and measurement of small molecule metabolites (<1700 Da) in complex biological matrices. This study sought to assess the current understanding and usage of metabolomics in laboratory medicine globally and evaluate the perception of its promise and future implementation. METHODS: A survey was conducted by the IFCC metabolomics working group that queried 400 professionals from 79 countries. Participants provided insights into their experience levels, knowledge, and usage of metabolomics approaches, along with detailing the applications and methodologies employed. RESULTS: Findings revealed a varying level of experience among respondents, with varying degrees of familiarity and utilization of metabolomics techniques. Targeted approaches dominated the field, particularly liquid chromatography coupled to a triple quadrupole mass spectrometer, with untargeted methods also receiving significant usage. Applications spanned clinical research, epidemiological studies, clinical diagnostics, patient monitoring, and prognostics across various medical domains, including metabolic diseases, endocrinology, oncology, cardiometabolic risk, neurodegeneration and clinical toxicology. CONCLUSIONS: Despite optimism for the future of clinical metabolomics, challenges such as technical complexity, standardization issues, and financial constraints remain significant hurdles. The study underscores the promising yet intricate landscape of metabolomics in clinical practice, emphasizing the need for continued efforts to overcome barriers and realize its full potential in patient care and precision medicine.

De novo NAD+ synthesis enhances mitochondrial function and improves health.

Nicotinamide adenine dinucleotide (NAD+) is a co-substrate for several enzymes, including the sirtuin family of NAD+-dependent protein deacylases. Beneficial effects of increased NAD+ levels and sirtuin activation on mitochondrial homeostasis, organismal metabolism and lifespan have been established across species. Here we show that α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of α-amino-ß-carboxymuconate-ε-semialdehyde in the de novo NAD+ synthesis pathway, controls cellular NAD+ levels via an evolutionarily conserved mechanism in Caenorhabditis elegans and mouse. Genetic and pharmacological inhibition of ACMSD boosts de novo NAD+ synthesis and sirtuin 1 activity, ultimately enhancing mitochondrial function. We also characterize two potent and selective inhibitors of ACMSD. Because expression of ACMSD is largely restricted to kidney and liver, these inhibitors may have therapeutic potential for protection of these tissues from injury. In summary, we identify ACMSD as a key modulator of cellular NAD+ levels, sirtuin activity and mitochondrial homeostasis in kidney and liver.

Omic-Scale High-Throughput Quantitative LC-MS/MS Approach for Circulatory Lipid Phenotyping in Clinical Research.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

Metabolomics meets lipidomics: Assessing the small molecule component of metabolism.

Metabolomics, including lipidomics, is emerging as a quantitative biology approach for the assessment of energy flow through metabolism and information flow through metabolic signaling; thus, providing novel insights into metabolism and its regulation, in health, healthy ageing and disease. In this forward-looking review we provide an overview on the origins of metabolomics, on its role in this postgenomic era of biochemistry and its application to investigate metabolite role and (bio)activity, from model systems to human population studies. We present the challenges inherent to this analytical science, and approaches and modes of analysis that are used to resolve, characterize and measure the infinite chemical diversity contained in the metabolome (including lipidome) of complex biological matrices. In the current outbreak of metabolic diseases such as cardiometabolic disorders, cancer and neurodegenerative diseases, metabolomics appears to be ideally situated for the investigation of disease pathophysiology from a metabolite perspective.

Sex-specific alterations in NAD+ metabolism in 3xTg Alzheimer's disease mouse brain assessed by quantitative targeted LC-MS.

Levels of nicotinamide adenine dinucleotide (NAD+) are known to decline with age and have been associated with impaired mitochondrial function leading to neurodegeneration, a key facet of Alzheimer's disease (AD). NAD+synthesis is sustained via tryptophan-kynurenine (Trp-Kyn) pathway as de novo synthesis route, and salvage pathways dependent on the availability of nicotinic acid and nicotinamide. While being currently investigated as a multifactorial disease with a strong metabolic component, AD remains without curative treatment and important sex differences were reported in relation to disease onset and progression. The aim of this study was to reveal the potential deregulation of NAD+metabolism in AD with the direct analysis of NAD+precursors in the mouse brain tissue (wild type (WT) versus triple transgenic (3xTg) AD), using a sex-balanced design. To this end, we developed a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which allowed for the measurement of the full spectrum of NAD+precursors and intermediates in all three pathways. In brain tissue of mice with developed AD symptoms, a decrease in kynurenine (Kyn) versus increase in kynurenic acid (KA) levels were observed in both sexes with a significantly higher increment of KA in males. These alterations in Trp-Kyn pathway might be a consequence of neuroinflammation and a compensatory production of neuroprotective kynurenic acid. In the NAD+ salvage pathway, significantly lower levels of nicotinamide mononucleotide (NMN) were measured in the AD brain of males and females. Depletion of NMN implies the deregulation of salvage pathway critical for maintaining optimal NAD+ levels and mitochondrial and neuronal function.

XCMS-MRM and METLIN-MRM: a cloud library and public resource for targeted analysis of small molecules.

We report XCMS-MRM and METLIN-MRM ( http://xcmsonline-mrm.scripps.edu/ and http://metlin.scripps.edu/ ), a cloud-based data-analysis platform and a public multiple-reaction monitoring (MRM) transition repository for small-molecule quantitative tandem mass spectrometry. This platform provides MRM transitions for more than 15,500 molecules and facilitates data sharing across different instruments and laboratories.

Merged Targeted Quantification and Untargeted Profiling for Comprehensive Assessment of Acylcarnitine and Amino Acid Metabolism.

Acylcarnitines and amino acids are key players in energy metabolism; however, analytical methods for comprehensive and straightforward quantitative profiling of these metabolites, without derivatization or use of ion-pairing agents, are lacking. We therefore developed a hydrophilic interaction chromatography (HILIC)-based high-resolution mass spectrometry (HRMS) method for the simultaneous quantification of acylcarnitines and amino acids in a single run, while taking advantage of HRMS data acquired in full-scan mode to screen for additional derivatives and other polar metabolites. A single-step metabolite extraction with internal standard mixture (in methanol) warranted high-throughput sample preparation whose applicability was demonstrated on a panel of human biofluids (i.e., blood plasma, CSF, and urine) and brain tissue. Method accuracy was within 90-106% of validated NIST reference plasma concentrations for the panel of measured amino acids. Amino acid and acylcarnitine extraction recoveries were 87-100% on average, depending on the concentration range spiked. The coefficient of variation (CV) was 1-10% and 1-25% for intra- and interday measurements, respectively, with the highest CVs for the metabolites at the limit of quantification, depending on the biofluid. Acylcarnitine and amino acid signatures or chemical composition barcodes of the different biofluids and human brain tissue were acquired and biofluid- and tissue-associated differences were discussed in the context of their respective physiological roles. Significant differences were observed in the amino acid profiles, whereas acylcarnitine composition did not show biofluid-characteristic or brain region-specific pattern. The retrospective exploration of full-scan all-ion-fragmentation data allowed us to extract the information on unsaturated and hydroxylated acylcarnitine species, amines, and purine and pyrimidine metabolites. This merged targeted and untargeted approach provides an innovative strategy for simultaneous and comprehensive assessment of acylcarnitine and amino acid metabolism in clinical research studies using relevant biofluids and tissue extracts.

Ammonium accumulation and chemokine decrease in culture media of Gcdh-/- 3D reaggregated brain cell cultures.

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain. We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh-/- mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys). Significant amounts of GA and 3-OHGA were detected in Gcdh-/- aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh-/- aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh-/- aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh-/- aggregates at DIV 14 and after exposure to Lys at DIV 8. This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh-/- brain cells. We described for the first time a decrease of chemokines in Gcdh-/- culture media which might contribute to brain cell injury in GA-I.

Autonomous Multimodal Metabolomics Data Integration for Comprehensive Pathway Analysis and Systems Biology.

Comprehensive metabolomic data can be achieved using multiple orthogonal separation and mass spectrometry (MS) analytical techniques. However, drawing biologically relevant conclusions from this data and combining it with additional layers of information collected by other omic technologies present a significant bioinformatic challenge. To address this, a data processing approach was designed to automate the comprehensive prediction of dysregulated metabolic pathways/networks from multiple data sources. The platform autonomously integrates multiple MS-based metabolomics data types without constraints due to different sample preparation/extraction, chromatographic separation, or MS detection method. This multimodal analysis streamlines the extraction of biological information from the metabolomics data as well as the contextualization within proteomics and transcriptomics data sets. As a proof of concept, this multimodal analysis approach was applied to a colorectal cancer (CRC) study, in which complementary liquid chromatography-mass spectrometry (LC-MS) data were combined with proteomic and transcriptomic data. Our approach provided a highly resolved overview of colon cancer metabolic dysregulation, with an average 17% increase of detected dysregulated metabolites per pathway and an increase in metabolic pathway prediction confidence. Moreover, 95% of the altered metabolic pathways matched with the dysregulated genes and proteins, providing additional validation at a systems level. The analysis platform is currently available via the XCMS Online ( XCMSOnline.scripps.edu ).

Data Streaming for Metabolomics: Accelerating Data Processing and Analysis from Days to Minutes.

The speed and throughput of analytical platforms has been a driving force in recent years in the "omics" technologies and while great strides have been accomplished in both chromatography and mass spectrometry, data analysis times have not benefited at the same pace. Even though personal computers have become more powerful, data transfer times still represent a bottleneck in data processing because of the increasingly complex data files and studies with a greater number of samples. To meet the demand of analyzing hundreds to thousands of samples within a given experiment, we have developed a data streaming platform, XCMS Stream, which capitalizes on the acquisition time to compress and stream recently acquired data files to data processing servers, mimicking just-in-time production strategies from the manufacturing industry. The utility of this XCMS Online-based technology is demonstrated here in the analysis of T cell metabolism and other large-scale metabolomic studies. A large scale example on a 1000 sample data set demonstrated a 10 000-fold time savings, reducing data analysis time from days to minutes. Further, XCMS Stream has the capability to increase the efficiency of downstream biochemical dependent data acquisition (BDDA) analysis by initiating data conversion and data processing on subsets of data acquired, expanding its application beyond data transfer to smart preliminary data decision-making prior to full acquisition.

Determining conserved metabolic biomarkers from a million database queries.

MOTIVATION: Metabolite databases provide a unique window into metabolome research allowing the most commonly searched biomarkers to be catalogued. Omic scale metabolite profiling, or metabolomics, is finding increased utility in biomarker discovery largely driven by improvements in analytical technologies and the concurrent developments in bioinformatics. However, the successful translation of biomarkers into clinical or biologically relevant indicators is limited. RESULTS: With the aim of improving the discovery of translatable metabolite biomarkers, we present search analytics for over one million METLIN metabolite database queries. The most common metabolites found in METLIN were cross-correlated against XCMS Online, the widely used cloud-based data processing and pathway analysis platform. Analysis of the METLIN and XCMS common metabolite data has two primary implications: these metabolites, might indicate a conserved metabolic response to stressors and, this data may be used to gauge the relative uniqueness of potential biomarkers. AVAILABILITY AND IMPLEMENTATION: METLIN can be accessed by logging on to: https://metlin.scripps.edu CONTACT: siuzdak@scripps.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Ammonium accumulation is a primary effect of 2-methylcitrate exposure in an in vitro model for brain damage in methylmalonic aciduria.

Using 3D organotypic rat brain cell cultures in aggregates we recently identified 2-methylcitrate (2-MCA) as the main toxic metabolite for developing brain cells in methylmalonic aciduria. Exposure to 2-MCA triggered morphological changes and apoptosis of brain cells. This was accompanied by increased ammonium and decreased glutamine levels. However, the sequence and causal relationship between these phenomena remained unclear. To understand the sequence and time course of pathogenic events, we exposed 3D rat brain cell aggregates to different concentrations of 2-MCA (0.1, 0.33 and 1.0mM) from day in vitro (DIV) 11 to 14. Aggregates were harvested at different time points from DIV 12 to 19. We compared the effects of a single dose of 1mM 2-MCA administered on DIV 11 to the effects of repeated doses of 1mM 2-MCA. Pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh were used to block apoptosis. Ammonium accumulation in the culture medium started within few hours after the first 2-MCA exposure. Morphological changes of the developing brain cells were already visible after 17h. The highest rate of cleaved caspase-3 was observed after 72h. A dose-response relationship was observed for all effects. Surprisingly, a single dose of 1mM 2-MCA was sufficient to induce all of the biochemical and morphological changes in this model. 2-MCA-induced ammonium accumulation and morphological changes were not prevented by concomitant treatment of the cultures with pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh: ammonium increased rapidly after a single 1mM 2-MCA administration even after apoptosis blockade. We conclude that following exposure to 2-MCA, ammonium production in brain cell cultures is an early phenomenon, preceding cell degeneration and apoptosis, and may actually be the cause of the other changes observed. The fact that a single dose of 1mM 2-MCA is sufficient to induce deleterious effects over several days highlights the potential damaging effects of even short-lasting metabolic decompensations in children affected by methylmalonic aciduria.

Thermal Degradation of Small Molecules: A Global Metabolomic Investigation.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 °C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online ( xcmsonline.scripps.edu ). The results showed that heating at an elevated temperature of 100 °C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 °C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 °C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.

Autonomous metabolomics for rapid metabolite identification in global profiling.

An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level.