bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒04‒25
twenty-four papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Mol Omics. 2021 Apr 20.
      Metabolic reprogramming is a hallmark of cancer, which is still far from being fully understood in colorectal cancer. In order to characterize the metabolic changes in colorectal cancer, we performed metabolomics analysis of paired colon tissues from colorectal cancer patients by using a liquid chromatography-mass spectrometry (LC-MS)-based method. Bioinformation analysis was used to define important metabolites and metabolic pathways, as well as the prognosis significance and expression levels of the key molecules. The results indicated that the metabolite phenotype in cancerous colon tissues was obviously different from their normal counterpart, and we identified a series of important metabolic changes in colorectal cancer, including decreased trends of glucose, citrate, serotonin, 5-hydroxytryptophol and 5-hydroxyindoleacetate, as well as increased trends of glutamate, glutathione, creatine, proline, lactate, fructose 1,6-bisphosphate, succinate, tryptophan, kynurenine and long chain acyl-carnitines. These metabolites are mainly implicated in energy metabolism, amino acid metabolism, glutathione metabolism and fatty acid metabolism. In addition, we found that the expression levels of several key molecules in these pathways were closely correlated with the prognosis of colorectal cancer patients. This study characterizes the metabolic profile in colorectal cancer tissues and provides more insightful understanding of the metabolic reprogramming of colorectal cancer.
  2. Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2019474118. [Epub ahead of print]118(17):
      Insulin and insulin-like growth factor 1 (IGF-1) receptors share many downstream signaling pathways but have unique biological effects. To define the molecular signals contributing to these distinct activities, we performed global phosphoproteomics on cells expressing either insulin receptor (IR), IGF-1 receptor (IGF1R), or chimeric IR-IGF1R receptors. We show that IR preferentially stimulates phosphorylations associated with mammalian target of rapamycin complex 1 (mTORC1) and Akt pathways, whereas IGF1R preferentially stimulates phosphorylations on proteins associated with the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases), and cell cycle progression. There were also major differences in the phosphoproteome between cells expressing IR versus IGF1R in the unstimulated state, including phosphorylation of proteins involved in membrane trafficking, chromatin remodeling, and cell cycle. In cells expressing chimeric IR-IGF1R receptors, these differences in signaling could be mapped to contributions of both the extra- and intracellular domains of these receptors. Thus, despite their high homology, IR and IGF1R preferentially regulate distinct networks of phosphorylation in both the basal and stimulated states, allowing for the unique effects of these hormones on organismal function.
    Keywords:  IGF-1 signaling; cellular signaling; insulin signaling; kinases; protein phosphorylation
  3. Mol Cell Proteomics. 2021 Apr 19. pii: S1535-9476(21)00055-4. [Epub ahead of print] 100082
      Reversed-phase high performance liquid chromatography (HPLC) is the most commonly applied peptide separation technique in mass spectrometry-based proteomics. Particle-packed capillary columns are predominantly used in nano-flow HPLC systems. Despite being the broadly applied standard for many years capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column packing with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as driving fluid, our system replaces the traditional setup of helium pressured packing bombs. By using 10x multiplexing, we have reduced the production time to just under 2 minutes for several 50 cm columns with 1.9 um particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters (ID) and length packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station.
    Keywords:  HPLC; LC-MS
  4. Front Oncol. 2021 ;11 579286
      The challenge of eradicating cancer is that cancer cells possess diverse mechanisms to protect themselves from clinical strategies. Recently, ferroptosis has been shown to exhibit appreciable anti-tumor activity that could be harnessed for cancer therapy in the future. Ferroptosis is an iron-dependent form of regulated cell death that is characterized by the oxidization of polyunsaturated fatty acids (PUFAs) and accumulation of lipid peroxides. Ferroptosis has been closely correlated with numerous biological processes, such as amino acid metabolism, glutathione metabolism, iron metabolism, and lipid metabolism, as well as key regulators including GPX4, FSP1, NRF2, and p53. Although ferroptosis could be involved in killing various cancer cells, multiple aspects of this phenomenon remain unresolved. In this review, we summarize the biochemistry and biology of ferroptosis in diverse cancers and discuss the potential mechanisms of ferroptosis, which might pave the way for guiding cancer therapeutics.
    Keywords:  amino acid metabolism; cancers; ferroptosis; iron metabolism; lipid metabolism
  5. Anal Biochem. 2021 Apr 02. pii: S0003-2697(21)00093-2. [Epub ahead of print]624 114192
      In addition to the altered amino acids in many cancer cells for their uncontrolled growth, targeted metabolomics in cell culture media could display a dynamic interaction between cancer cells and their micro-environments. Methodology for cell culture medium samples is different from that of cell lysates on sampling points, calculation and statistical analysis. Targeted profiling method of 40 amino acid and derivatives was validated and performed on cell culture medium samples from cell lines of HCC 1806 (breast cancer cell) and MCF-10A (normal breast epithelial cell). Different from the common up-regulation of amino acids in cancer cell lysates, significantly increased uptake (>2.5-fold, VIP>1 and p < 0.001) of branched amino acids was observed in the cell culture media from the breast cancer cells while acetylmethionine, cysteine-glutathione, glutathione, cysteine and glutamic acid were excreted significantly more by the cancer cells to their media. The characteristic metabolic changes of amino acid and derivatives in the cell culture media provide a dynamic portrayal for the interaction of the breast cancer cells, normal breast cells with their micro-environments, which helps to understand the underlying proliferation mechanism of breast cancer cells.
    Keywords:  Amino acid and derivatives; Breast cancer cell; Cell culture medium; HILIC-MS/MS
  6. Expert Rev Proteomics. 2021 Apr 19. 1-8
      Elucidating the dysregulated metabolic pathways in cancer cells and their relevance to cisplatin resistance could yield new insights into cancer therapy. We previously reported that eight metabolites involved in the tricarboxylic acid (TCA) cycle and glutamine metabolism were associated with platinum-based chemotherapy efficacy in human lung cancer. Here, we investigated the metabolic differences upon cisplatin treatment in lung cancer in vitro and in vivo. A simple and partially validated standard addition method was applied for the quantification of five metabolites involved in the TCA cycle and glutamine metabolism using amide hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The present study investigated the levels of these biomarkers in A549 cells and the cisplatin-resistant A549-DDP cells, as well as in the plasma before and after cisplatin treatment in A549 xenograft mice. Levels of five metabolites, including 2-hydroxyglutaric acid (2-HG), α-ketoglutarate (α-KG), succinate, glutamine, and glutamate, showed a decreasing trend in A549-DDP cells. In addition, 2-HG and glutamine were the most significantly altered metabolites in cisplatin-treated A549 xenograft mice. These data indicate that the TCA cycle and glutamine metabolism play important roles in cisplatin-based chemotherapy resistance in lung cancer. Our results provide a new angle for exploring the molecular mechanisms underlying cisplatin resistance.
    Keywords:  Cisplatin resistance; HILIC-MS/MS; TCA cycle; glutamine metabolism; lung cancer
  7. Mol Metab. 2021 Apr 20. pii: S2212-8778(21)00083-1. [Epub ahead of print] 101238
      BACKGROUND: Non-alcoholic fatty liver disease, or as recently proposed 'metabolic-associated fatty liver disease' (MAFLD), is characterized by pathological accumulation of triglycerides and other lipids in hepatocytes. This common disease can progress from simple steatosis to steatohepatitis, and eventually end-stage liver diseases. MAFLD is closely related to disturbances in systemic energy metabolism, including insulin resistance and atherogenic dyslipidemia.SCOPE OF REVIEW: The liver is the central organ in lipid metabolism by secreting very low density lipoproteins (VLDL) and, on the other hand, by internalizing fatty acids and lipoproteins. This review article discusses recent research addressing hepatic lipid synthesis, VLDL production and lipoprotein internalization as well as the lipid exchange between adipose tissue and liver in the context of MAFLD.
    MAJOR CONCLUSIONS: Liver steatosis in MAFLD is triggered by excessive hepatic triglyceride synthesis utilizing fatty acids derived from white adipose tissue (WAT), de novo lipogenesis (DNL) and endocytosed remnants of triglyceride-rich lipoproteins. In consequence of high hepatic lipid content VLDL secretion is enhanced, which is the primary cause of complex dyslipidemia typical for subjects with MAFLD. Interventions reducing VLDL secretory capacity attenuate dyslipidemia while they exacerbate MAFLD, indicating that the balance of lipid storage versus secretion in hepatocytes is a critical parameter determining disease outcome. Proof of concept studies have shown that promoting lipid storage and energy combustion in adipose tissues reduces hepatic lipid load and thus ameliorates MAFLD. Moreover, hepatocellular triglyceride synthesis from DNL and WAT-derived fatty acids can be targeted to treat MAFLD. However, more research is needed to understand how individual transporters, enzymes and their isoforms affect steatosis and dyslipidemia in vivo, and whether these two aspects of MAFLD can be selectively treated. Processing of cholesterol-enriched lipoproteins appears less important for steatosis. It may, however, modulate inflammation and consequently MAFLD progression.
    Keywords:  NAFLD; adipose tissue; cholesterol; de novo lipogenesis; lipoprotein; lipotoxicity; liver; triglycerides
  8. J Am Soc Mass Spectrom. 2021 Apr 05.
      The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.
    Keywords:  H3 variants; cell cycle; histone modifications; middle-down; quantitative proteomics
  9. Cell Metab. 2021 Apr 16. pii: S1550-4131(21)00166-2. [Epub ahead of print]
      Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.
    Keywords:  FGF21; GCN2; body mass index; branched-chain amino acids; diabetes; insulin resistance; isoleucine; mTORC1; obesity; valine
  10. Free Radic Res. 2021 Apr 19. 1-12
      Cellular and organismal redox imbalance leading to the accumulation of reactive oxygen species significantly enhances lipid peroxidation (LPO). LPO is relatively well studied for phospholipid membranes and to some extent for circulating lipoproteins. However, it is rarely addressed for intracellular lipid droplets (LDs). Here we optimized an in vitro model system to investigate oxidizability of different lipid classes within artificial LDs (aLDs). To this end, aLDs were reconstructed using differential centrifugation and characterized by a variety of analytical methods. Influence of different lipid compositions on aLDs size was studied and showed opposing effects of unsaturated phospholipids (PLs), triacyclglycerols (TAGs) and cholesteryl esters (CEs). To address aLDs oxidizability, the LPO sensitive ratiometric probe BODIPY-C11 was infused into aLDs, and lipid peroxidation kinetics, upon LPO activation either by copper/ascorbate or 2,2'-azobis(2-methylpropionamidine), was followed up by fluorescence spectroscopy. Generated lipid peroxidation products were additionally identified and relatively quantified by high-resolution LC-MS/MS. It was demonstrated that lipid composition is detrimental to aLD's oxidation sensitivity. Increasing unsaturation levels in the PL monolayer or the TAG core increases oxidation sensitivity, whereas the presence of CEs in the LD core has a dual effect depending on the acylated fatty acid. Moreover, not only the total level of lipid unsaturation, but also the ratio between different lipid species was shown to play a significant role in LPO propagation. This shows that the lipid composition of aLD's determines their sensitivity to LPO. As LDs lipidome reflects and is dynamically influenced by cellular and organismal metabolic status, our findings provide an important observation linking LD lipid composition and their redox sensitivity.
    Keywords:  Lipid peroxidation; lipid droplet; mass spectrometry; oxidized cholesteryl ester; oxidized triacylglycerol
  11. Amino Acids. 2021 Apr 05.
      Recent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.
    Keywords:  Apoptosis; Autophagy; Collagen metabolism; Prolidase; Proline; Proline dehydrogenase/Proline oxidase; Signalling
  12. Expert Rev Proteomics. 2021 Apr 20. 1-12
      Introduction: Cancer cell lines (CCLs) have been a major resource for cancer research. Over the past couple of decades, they have been instrumental in omic profiling method development and as model systems to generate new knowledge in cell and cancer biology. More recently, with the increasing amount of genomic, transcriptomic and proteomic data being generated in hundreds of CCLs, there is growing potential for integrative proteogenomic data analyses to be performed.Areas covered: In this review, we first describe the most commonly used proteome profiling methods in CCLs. We then discuss how these proteomics data can be integrated with genomics data for proteogenomics analyses. Finally, we highlight some of the recent biological discoveries that have arisen from proteogenomics analyses of CCLs.Expert opinion: Protegeonomics analyses of CCLs have so far enabled the discovery of novel proteins and proteoforms. It has also improved our understanding of biological processes including post-transcriptional regulation of protein abundance and the presentation of antigens by major histocompatibility complex alleles. With proteomics data to be generated in hundreds to thousands of CCLs in coming years, there will be further potential for large-scale proteogenomics analyses and data integration with the phenotypically well-characterized CCLs.
    Keywords:  Cancer cell lines; mass spectrometry; protein quantification; proteogenomics
  13. J Agric Food Chem. 2021 Apr 23.
      In addition to their important role in fat digestion, bile acids are increasingly being used as markers for various diseases. The large diversity of bile acids results from the conversion of primary and conjugated bile acids into secondary bile acids by deconjugation and dehydroxylation reactions mediated by the intestinal microbiota. Here, we describe a fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for absolute quantitation of 45 bile acids in human or mouse feces in combination with a simple workup and extraction procedure. Method validation outlined excellent limits of detection and quantitation, linearity, selectivity, recovery, extraction loss, and precision. To investigate the connection between microbiome alterations and bile acid metabolism, the method was applied on a Crohn's disease study including patients with histologically documented active disease or remission as well as on a model using humanized mice. As the complex mechanism including genetic and environmental factors leading to the development of Crohn's disease is so far not completely understood, the study investigates the microbial metabolism of bile acids and the potential use of bile acid profiles to predict disease state.
    Keywords:  Crohn’s disease; UHPLC-ESI-LC-MS/MS; bile acids; method validation; targeted metabolite profiling
  14. Science. 2021 Apr 22. pii: eabd5491. [Epub ahead of print]
      The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.
  15. Food Microbiol. 2021 Sep;pii: S0740-0020(21)00064-2. [Epub ahead of print]98 103799
      Vinegar is elaborated using a semi-continuous submerged culture of a complex microbiota of acetic acid bacteria. The genus Komagataeibacter provides much of the proteins of the metaproteome, being K. europaeus the main species working in this environment. In this work, the protein profile of the vinegar microbiota, obtained by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples from different cycle times of an acetification process using an alcohol medium, has been used to describe the functional metaproteome throughout the process. The analysis was focused on Komagataeibacter species which supplied about 90% of the metaproteome and particularly K. europaeus which accounts for more than 70%. According to these results, the natural behaviour of a microbial community in vinegar has been predicted at a quantitative proteomic level. The results revealed that most of the identified proteins involved in the metabolism of amino acids, biosynthesis of proteins, and energy production related-metabolic pathways increased their expression throughout the cycle loading phase and afterwards experimented a decrease coming into play other proteins acting against acetic acid stress. These findings may facilitate a better understanding of the microbiota's role and contributing to obtain a quality product.
    Keywords:  Alcohol vinegar; Komagataeibacter europaeus; LC-MS/MS; Quantitative proteomics; Submerged culture
  16. J Mol Med (Berl). 2021 Apr 21.
      Chronic cortisol excess induces several alterations on protein, lipid and carbohydrate metabolism resembling those found in the metabolic syndrome. However, patients exposed to prolonged high levels of cortisol in Cushing syndrome (CS) present exceeding cardiometabolic alterations not reflected by conventional biomarkers. Using 3 ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) platforms, we aimed to characterise the serum metabolome of 25 patients with active endogenous CS and 25 control subjects matched by propensity score (sex, BMI, diabetes mellitus type 2 (T2D), high blood pressure (HBP) and dyslipidaemia) to search for potential disease-specific biomarkers and pathways associated to the clinical comorbidities. A total of 93 metabolites were significantly altered in patients with CS. Increased levels of sulfur amino acids (AA), triacylglycerols, glycerophospholipids, ceramides and cholesteryl esters were observed. Contrarily, concentrations of essential and non-essential AA, polyunsaturated fatty acids, conjugated bile acids and second messenger glycerolipids were decreased. Twenty-four-hour urinary free cortisol (24h-UFC) independently determined the concentration of 21 lipids and 4 AA. A metabolic signature composed by 10 AA and 10 lipid metabolites presented an AUC-ROC of 95% for the classification of CS patients. Through differential network analysis, 152 aberrant associations between metabolites involved in the Lands cycle and Kennedy pathway were identified. Our data indicates that chronic hypercortisolemia confers a unique lipidomic signature and several alterations in numerous AA even when compared to patients with similar metabolic comorbidities providing novel insights of the increased cardiometabolic burden of CS. KEY MESSAGES: • Cortisol excess induces metabolic alterations beyond conventional biomarkers. • The hypercortisolism extent determines the concentration of 21 lipids and 5 aa. • Cortisol excess confers a unique metabolic signature of 20 metabolites. • Kennedy and Lands cycle are profoundly disturbed by cortisol excess.
    Keywords:  Biomarkers; Cortisol; Cushing syndrome; Lipidomics; Metabolomics
  17. Metabolomics. 2021 Apr 23. 17(5): 44
      INTRODUCTION: Manganese is important for the endocarditis pathogen Streptococcus sanguinis. Little is known about why manganese is required for virulence or how it impacts the metabolome of streptococci.OBJECTIVES: We applied untargeted metabolomics to cells and media to understand temporal changes resulting from manganese depletion.
    METHODS: EDTA was added to a S. sanguinis manganese-transporter mutant in aerobic fermentor conditions. Cell and media samples were collected pre- and post-EDTA treatment. Metabolomics data were generated using positive and negative modes of data acquisition on an LC-MS/MS system. Data were subjected to statistical processing using MetaboAnalyst and time-course analysis using Short Time series Expression Miner (STEM). Recombinant enzymes were assayed for metal dependence.
    RESULTS: We observed quantitative changes in 534 and 422 metabolites in cells and media, respectively, after EDTA addition. The 173 cellular metabolites identified as significantly different indicated enrichment of purine and pyrimidine metabolism. Further multivariate analysis revealed that the top 15 cellular metabolites belonged primarily to lipids and redox metabolites. The STEM analysis revealed global changes in cells and media in comparable metabolic pathways. Glycolytic intermediates such as fructose-1,6-bisphosphate increased, suggesting that enzymes that utilize them require manganese for activity or expression. Recombinant enzymes were confirmed to utilize manganese in vitro. Nucleosides accumulated, possibly due to a blockage in conversion to nucleobases resulting from manganese-dependent regulation.
    CONCLUSION: Differential analysis of metabolites revealed the activation of a number of metabolic pathways in response to manganese depletion, many of which are connected to carbon catabolite repression.
    Keywords:  Endocarditis; Manganese; Metabolomics; Multivariate; Time-course
  18. J Mass Spectrom. 2021 Mar 10. 56(5): e4718
      The use of ion mobility separations (IMSs) in metabolomics approaches has started to be deeply explored in the last years. In this work, the use of liquid chromatography (LC) coupled to IMS-quadrupole time-of-flight mass spectrometry (QTOF MS) has been evaluated in a metabolomics experiments using single injection of the samples. IMS has allowed obtaining cleaner fragmentation spectra, of nearly tandem MS quality, in data-independent acquisition mode. This is much useful in this research area as a second injection, generally applied in LC-QTOF MS workflows to obtain tandem mass spectra, is not necessary, saving time and evading possible compound degradation. As a case study, the smoke produced after combustion of herbal blends used to spray synthetic cannabinoids has been selected as study matrix. The smoke components were trapped in carbon cartridges, desorbed and analyzed by LC-IMS-QTOF MS using different separation mechanisms (reversed phase and HILIC) and acquiring in both positive and negative mode to widen the chemical domain. Partial Least Squares-Discriminant Analysis highlighted several compounds, and ratio between N-Isopropyl-3-(isoquinolinyl)-2-propen-1-amine and quinoline allowed differentiating between tobacco and herbal products. These two compounds were tentatively identified using the cleaner fragmentation spectra from a single injection in the IMS-QTOF MS, with additional confidence obtained by retention time (Rt) and collisional cross section (CCS) prediction using artificial neural networks. Data from this work show that LC-IMS-QTOF is an efficient technique in untargeted metabolomics, avoiding re-injection of the samples for elucidation purposes. In addition, the prediction models for Rt and CCS resulted of help in the elucidation process of potential biomarkers.
    Keywords:  herbal blends smoke; high resolution mass spectrometry; in-silico prediction; ion mobility; omics approaches
  19. Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2018229118. [Epub ahead of print]118(17):
      Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.
    Keywords:  GCN2; metabolism; myeloma; proteasome; proteostasis
  20. J Hepatol. 2021 Apr 19. pii: S0168-8278(21)00248-8. [Epub ahead of print]
      BACKGROUND: Obesity often leads to nonalcoholic fatty liver disease (NAFLD), which can progress from simple steatosis (nonalcoholic fatty liver (NAFL)) to nonalcoholic steatohepatitis (NASH). The accumulation of certain lipid subtypes is linked with worsening metabolic and liver disease, however, specific changes during progression from No-NAFL to NAFL then NASH are unresolved. Here, we characterise the liver, adipose tissue and plasma lipidome of worsening NAFLD in obesity, and evaluate the utility of plasma lipids as biomarkers of NAFLD.METHODS: Venous blood, liver, visceral and subcutaneous adipose tissue samples were obtained from 181 patients undergoing bariatric surgery. NAFLD severity was assessed histologically. Lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry.
    RESULTS: The liver lipidome showed substantial changes with increasing steatosis, with increased triacylglycerols, diacylglycerols and sphingolipids including ceramide, dihydroceramide, hexosyl-ceramide and GM3 ganglioside species. These lipid species were also increased in plasma with increasing hepatic steatosis and showed strong correlations with liver lipids. Adipose tissue lipidomes showed no correlation to NAFLD. There were no significant changes in liver lipids with NASH compared to NAFL. Addition of plasma lipid variables to routine markers yielded significant improvements in diagnostic accuracy for NASH (AUROC 0.667 vs 0.785, p=0.025).
    CONCLUSION: Overall, these data provide a detailed description of the lipidomic changes with worsening NAFLD, showing significant changes with steatosis but no additional changes with NASH. Alterations in the liver lipidome are paralleled by similar changes in plasma. Further investigation is warranted into the potential utility of plasma lipids as non-invasive biomarkers of NAFLD in obesity.
    LAY SUMMARY: Nonalcoholic fatty liver disease (NAFLD) is characterised by distinct changes in the liver lipidome with steatosis. The development of nonalcoholic steatohepatitis (NASH) does not result in further change in lipidome. Lipids within body fat does not appear to influence the lipid profile of liver or blood. Changes in liver lipids are paralleled by changes in blood lipids. This has potential to be developed into a non-invasive biomarker for NAFLD.
    Keywords:  Adipose tissue; Ceramides; Lipid metabolism; Lipid species; Lipidomics; Nonalcoholic fatty liver disease; Obesity; Triglycerides
  21. Nat Commun. 2021 Apr 23. 12(1): 2398
      Arginine plays diverse roles in cellular physiology. As a semi-essential amino acid, arginine deprivation has been used to target cancers with arginine synthesis deficiency. Arginine-deprived cancer cells exhibit mitochondrial dysfunction, transcriptional reprogramming and eventual cell death. In this study, we show in prostate cancer cells that arginine acts as an epigenetic regulator to modulate histone acetylation, leading to global upregulation of nuclear-encoded oxidative phosphorylation (OXPHOS) genes. TEAD4 is retained in the nucleus by arginine, enhancing its recruitment to the promoter/enhancer regions of OXPHOS genes and mediating coordinated upregulation in a YAP1-independent but mTOR-dependent manner. Arginine also activates the expression of lysine acetyl-transferases and increases overall levels of acetylated histones and acetyl-CoA, facilitating TEAD4 recruitment. Silencing of TEAD4 suppresses OXPHOS functions and prostate cancer cell growth in vitro and in vivo. Given the strong correlation of TEAD4 expression and prostate carcinogenesis, targeting TEAD4 may be beneficially used to enhance arginine-deprivation therapy and prostate cancer therapy.
  22. J Proteome Res. 2021 Apr 17.
      Osteoarthritis (OA) is a multifactorial pathology and comprises a wide range of distinct phenotypes. In this context, the characterization of the different molecular profiles associated with each phenotype can improve the classification of OA. In particular, OA can coexist with type 2 diabetes mellitus (T2DM). This study investigates lipidomic and proteomic differences between human OA/T2DM- and OA/T2DM+ cartilage through a multimodal mass spectrometry approach. Human cartilage samples were obtained after total knee replacement from OA/T2DM- and OA/T2DM+ patients. Label-free proteomics was employed to study differences in protein abundance and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for spatially resolved-lipid analysis. Label-free proteomic analysis showed differences between OA/T2DM- and OA/T2DM+ phenotypes in several metabolic pathways such as lipid regulation. Interestingly, phospholipase A2 protein was found increased within the OA/T2DM+ cohort. In addition, MALDI-MSI experiments revealed that phosphatidylcholine and sphingomyelin species were characteristic of the OA/T2DM- group, whereas lysolipids were more characteristic of the OA/T2DM+ phenotype. The data also pointed out differences in phospholipid content between superficial and deep layers of the cartilage. Our study shows distinctively different lipid and protein profiles between OA/T2DM- and OA/T2DM+ human cartilage, demonstrating the importance of subclassification of the OA disease for better personalized treatments.
    Keywords:  MALDI-MSI; cartilage; diabetes; label-free proteomics; osteoarthritis; spatially resolved-lipid analysis
  23. Mol Cell Proteomics. 2021 Apr 19. pii: S1535-9476(21)00056-6. [Epub ahead of print] 100083
      Untargeted mass spectrometry (MS)-based proteomics provides a powerful platform for protein biomarker discovery, but clinical translation depends on the selection of a small number of proteins for downstream verification and validation. Due to the small sample size of typical discovery studies, protein markers identified from discovery data may not be generalizable to independent datasets. In addition, a good protein marker identified using a discovery platform may be difficult to implement in verification and validation platforms. Moreover, although multi-omics characterization is being increasingly used in discovery cohort studies, there is no existing method for multi-omics-facilitated protein biomarker selection. Here, we present ProMS, a computational algorithm for protein marker selection. The algorithm is based on the hypothesis that a phenotype is characterized by a few underlying biological functions, each manifested by a group of co-expressed proteins. A weighted k- medoids clustering algorithm is applied to all univariately informative proteins to identify both co-expressed protein clusters and a representative protein for each cluster as markers. In two clinically-important classification problems, ProMS shows superior performance compared with existing feature selection methods. ProMS can be extended to the multi-omics setting (ProMS_mo) through a constrained weighted k-medoids clustering algorithm, and the protein panels selected by ProMS_mo show improved performance on independent test data compared with ProMS. In addition to superior performance, ProMS and ProMS_mo also have two unique strengths. First, the feature clusters enable functional interpretation of the selected protein markers. Second, the feature clusters provide an opportunity to select replacement protein markers, facilitating a robust transition to the verification and validation platforms. In summary, this study provides a unified and effective computational framework for selecting protein biomarkers using proteomics or multi-omics data. The software implementation is publicly available at
  24. Anal Chem. 2021 Apr 21.
      Polar phosphorylated metabolites are involved in a variety of biological processes and play vital roles in energetic metabolism, cofactor regeneration, and nucleic acid synthesis. However, it is often challenging to interrogate polar phosphorylated metabolites and compounds from biological samples. Liquid chromatography-mass spectrometry (LC/MS) now plays a central role in metabolomic studies. However, LC/MS-based approaches have been hampered by the issues of the low ionization efficiencies, low in vivo concentrations, and less chemical stability of polar phosphorylated metabolites. In this work, we synthesized paired reagents of light and heavy isotopomers, 2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (DMPI) and d3-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (d3-DMPI). The paired reagents of DMPI and d3-DMPI carry diazo groups that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites under mild conditions. As a proof of concept, we found that the transfer of the indole heterocycle group from DMPI/d3-DMPI to ribonucleotides led to the significant increase of ionization efficiencies of ribonucleotides during LC/MS analysis. The detection sensitivities of these ribonucleotides increased by 25-1137-fold upon DMPI tagging with the limits of detection (LODs) being between 7 and 150 amol. With the developed method, we achieved the determination of all the 12 ribonucleotides from a single mammalian cell and from a single stamen of Arabidopsis thaliana. The method provides a valuable tool to investigate the dynamic changes of polar phosphorylated metabolites in a single cell under particular conditions.