bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒09‒22
sixteen papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

  1. Metabolites. 2019 Sep 17. pii: E187. [Epub ahead of print]9(9):
      Technological advancements have permitted the development of innovative multiplexing strategies for data independent acquisition (DIA) mass spectrometry (MS). Software solutions and extensive compound libraries facilitate the efficient analysis of MS1 data, regardless of the analytical platform. However, the development of comparable tools for DIA data analysis has significantly lagged. This research introduces an update to the former MetaboList R package and a workflow for full-scan MS1 and MS/MS DIA processing of metabolomic data from multiplexed liquid chromatography high-resolution mass spectrometry (LC-HRMS) experiments. When compared to the former version, new functions have been added to address isolated MS1 and MS/MS workflows, processing of MS/MS data from stepped collision energies, performance scoring of metabolite annotations, and batch job analysis were incorporated into the update. The flexibility and efficiency of this strategy were assessed through the study of the metabolite profiles of human urine, leukemia cell culture, and medium samples analyzed by either liquid chromatography quadrupole time-of-flight (q-TOF) or quadrupole orbital (q-Orbitrap) instruments. This open-source alternative was designed to promote global metabolomic strategies based on recursive retrospective research of multiplexed DIA analysis.
    Keywords:  R programming; R-MetaboList 2; all ion fragmentation; data-independent acquisition; full-scan MS/MS processing; liquid chromatography high-resolution mass spectrometry; metabolomics; targeted analysis; untargeted analysis
  2. Metabolites. 2019 Sep 14. pii: E184. [Epub ahead of print]9(9):
      Several small molecule biomarkers have been reported in the literature for prediction and diagnosis of (pre)diabetes, its co-morbidities, and complications. Here, we report the development and validation of a novel, quantitative method for the determination of a selected panel of 34 metabolite biomarkers from human plasma. We selected a panel of metabolites indicative of various clinically-relevant pathogenic stages of diabetes. We combined these candidate biomarkers into a single ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and optimized it, prioritizing simplicity of sample preparation and time needed for analysis, enabling high-throughput analysis in clinical laboratory settings. We validated the method in terms of limits of detection (LOD) and quantitation (LOQ), linearity (R2), and intra- and inter-day repeatability of each metabolite. The method's performance was demonstrated in the analysis of selected samples from a diabetes cohort study. Metabolite levels were associated with clinical measurements and kidney complications in type 1 diabetes (T1D) patients. Specifically, both amino acids and amino acid-related analytes, as well as specific bile acids, were associated with macro-albuminuria. Additionally, specific bile acids were associated with glycemic control, anti-hypertensive medication, statin medication, and clinical lipid measurements. The developed analytical method is suitable for robust determination of selected plasma metabolites in the diabetes clinic.
    Keywords:  clinical diagnostics; diabetes; mass spectrometry; metabolomics
  3. Trends Analyt Chem. 2019 Nov;pii: 115644. [Epub ahead of print]120
      Mass spectrometry (MS)-based top-down proteomics characterizes complex proteomes at the intact proteoform level and provides an accurate picture of protein isoforms and protein post-translational modifications in the cell. The progress of top-down proteomics requires novel analytical tools with high peak capacity for proteoform separation and high sensitivity for proteoform detection. The requirements have made capillary zone electrophoresis (CZE)-MS an attractive approach for advancing large-scale top-down proteomics. CZE has achieved a peak capacity of 300 for separation of complex proteoform mixtures. CZE-MS has shown drastically better sensitivity than commonly used reversed-phase liquid chromatography (RPLC)-MS for proteoform detection. The advanced CZE-MS identified 6,000 proteoforms of nearly 1,000 proteoform families from a complex proteome sample, which represents one of the largest top-down proteomic datasets so far. In this review, we focus on the recent progress in CZE-MS-based top-down proteomics and provide our perspectives about its future directions.
    Keywords:  capillary zone electrophoresis; mass spectrometry; post-translational modification; protein complex; proteoform; top-down proteomics
  4. Nat Rev Cancer. 2019 Sep 17.
      The way cancer cells utilize nutrients to support their growth and proliferation is determined by cancer cell-intrinsic and cancer cell-extrinsic factors, including interactions with the environment. These interactions can define therapeutic vulnerabilities and impact the effectiveness of cancer therapy. Diet-mediated changes in whole-body metabolism and systemic nutrient availability can affect the environment that cancer cells are exposed to within tumours, and a better understanding of how diet modulates nutrient availability and utilization by cancer cells is needed. How diet impacts cancer outcomes is also of great interest to patients, yet clear evidence for how diet interacts with therapy and impacts tumour growth is lacking. Here we propose an experimental framework to probe the connections between diet and cancer metabolism. We examine how dietary factors may affect tumour growth by altering the access to and utilization of nutrients by cancer cells. Our growing understanding of how certain cancer types respond to various diets, how diet impacts cancer cell metabolism to mediate these responses and whether dietary interventions may constitute new therapeutic opportunities will begin to provide guidance on how best to use diet and nutrition to manage cancer in patients.
  5. Bioorg Chem. 2019 Sep 07. pii: S0045-2068(19)30436-5. [Epub ahead of print]92 103263
      α-Methylacyl-CoA racemase (AMACR; P504S) catalyses an essential step in the degradation of branched-chain fatty acids and the activation of ibuprofen and related drugs. AMACR has gained much attention as a drug target and biomarker, since it is found at elevated levels in prostate cancer and several other cancers. Herein, we report the synthesis of 2-(phenylthio)propanoyl-CoA derivatives which provided potent AMACR inhibitory activity (IC50 = 22-100 nM), as measured by the AMACR colorimetric activity assay. Inhibitor potency positively correlates with calculated logP, although 2-(3-benzyloxyphenylthio)propanoyl-CoA and 2-(4-(2-methylpropoxy)phenylthio)propanoyl-CoA were more potent than predicted by this parameter. Subsequently, carboxylic acid precursors were evaluated against androgen-dependent LnCaP prostate cancer cells and androgen-independent Du145 and PC3 prostate cancer cells using the MTS assay. All tested precursor acids showed inhibitory activity against LnCaP, Du145 and PC3 cells at 500 µM, but lacked activity at 100 µM. This is the first extensive structure-activity relationship study on the influence of side-chain interactions on the potency of novel rationally designed AMACR inhibitors.
    Keywords:  Branched-chain fatty acid metabolism; Castrate-resistant prostate cancer (CRPC); Drug lipophilicity; Enzyme inhibitors; Ibuprofen; Mixed competitive inhibition; Rational drug design; Structure-activity relationships; α-Methylacyl-CoA racemase (AMACR, P504S)
  6. Cell Syst. 2019 Sep 06. pii: S2405-4712(19)30274-1. [Epub ahead of print]
      Aberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.
    Keywords:  SRM; T-loop phosphorylation; cancer; kinase; kinase activity; phosphoproteomics; proteomics; signaling; targeted mass spectrometry
  7. Cancer Cell. 2019 Sep 16. pii: S1535-6108(19)30371-X. [Epub ahead of print]36(3): 319-336.e7
      The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) β-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.
    Keywords:  CRC intrinsic subtypes (CRIS); NOTCH1; TGF-β; colorectal cancer (CRC); consensus molecular subtype (CMS); metastasis; molecular subtyping; neutrophils; serrated CRC; tumor microenviroment (TME)
  8. Cell Rep. 2019 Sep 17. pii: S2211-1247(19)31074-5. [Epub ahead of print]28(12): 3011-3021.e4
      T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation.
    Keywords:  T cell activation; T cells; alanine; metabolism; protein synthesis
  9. Mol Cell Oncol. 2019 ;6(5): e1536843
      Cancer cells rely on glutamine to fuel mitochondria, however it remains unclear whether this is needed for bioenergetic or biosynthetic pathways. Our study suggests that an essential function of mitochondrial glutamine metabolism is to provide aspartate to the cytosol where it can be used for nucleotide and protein synthesis.
    Keywords:  AGC1; Aralar; CB-839; Glutamine; Slc25a12; TCA cycle; aspartate; aspartate-glutamate carrier; cancer metabolism; mitochondrial transporters; targeting metabolism
  10. J Pharm Biomed Anal. 2019 Sep 10. pii: S0731-7085(19)31150-1. [Epub ahead of print]177 112871
      Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000 nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6 nM due to low accuracy at 2 nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80 mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.
    Keywords:  AZ5104; Human plasm; LC–MS/MS; Non-small cell lung carcinoma; Osimertinib; Thiol conjugates
  11. EMBO J. 2019 Sep 16. e102147
      L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.
    Keywords:  L-asparaginase; SLC1A3; aspartate/glutamate; genome-wide CRISPR screen; solid tumors
  12. Am J Physiol Endocrinol Metab. 2019 Sep 17.
      Insulin resistance has wide-ranging effects on metabolism but there are knowledge gaps regarding the tissue origins of systemic metabolite patterns, and how patterns are altered by fitness and metabolic health. To address these questions, plasma metabolite patterns were determined every 5 min during exercise (30 min, ~45% of V̇O2peak, ~63 W) and recovery in overnight-fasted sedentary, obese, insulin resistant women under controlled conditions of diet and physical activity. We hypothesized that improved fitness and insulin sensitivity following a ~14 wk training and weight loss intervention would lead to fixed workload plasma metabolomics signatures reflective of metabolic health and muscle metabolism. Pattern analysis over the first 15 min of exercise-regardless of pre- vs. post-intervention status-highlighted anticipated increases in fatty acid tissue uptake and oxidation (e.g., reduced long-chain fatty acids), diminution of non-oxidative fates of glucose (e.g., lowered sorbitol-pathway metabolites and glycerol-3-galactoside [possible glycerolipid synthesis metabolite]), and enhanced tissue amino acid use (e.g., drops in amino acids; modest increase in urea). A novel observation was that exercise significantly increased several xenometabolites ("non-self" molecules, from microbes or foods), including benzoic acid/salicylic acid/salicylaldehyde, hexadecanol/octadecanol/dodecanol, and chlorogenic acid. In addition, many non-annotated metabolites changed with exercise. Although exercise itself strongly impacted the global metabolome, there were surprisingly few intervention-associated differences despite marked improvements in insulin sensitivity, fitness, and adiposity. These results, and previously-reported plasma acylcarnitine profiles, support the principle that most metabolic changes during sub-maximal aerobic exercise are closely tethered to absolute ATP turnover rate (workload), regardless of fitness or metabolic health status.
    Keywords:  cystine; muscle fatigue; succinate; taurine; xenobiotic
  13. Sci Rep. 2019 Sep 20. 9(1): 13613
      Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.
  14. Theranostics. 2019 ;9(19): 5478-5496
      Inhibiting STAT3 signaling reduces tumor progression, metastasis and chemoresistance, however the precise molecular mechanism has not been fully delineated in ovarian cancer.METHODS: In this study, we generated STAT3 knockout (KO) ovarian cancer cell lines. Effects of STAT3 KO on cell proliferation, migration and spheroid formation were assessed in vitro and effects on in vivo tumor growth were tested using several tumor xenograft models. We used multi-omic genome-wide profiling to identify multi-level (Bru-Seq, RNA-Seq, and MS Proteomic) expression signatures of STAT3 KO ovarian cancer cells.
    RESULTS: We observed that deletion of STAT3 blocked cell proliferation and migration in vitro and suppressed tumor growth in mice. Deletion of STAT3 transcriptionally suppressed key genes involved in EMT, cell cycle progression, E2F signaling, and altered stemness markers. Notably, KO of STAT3 resulted in modulation of the expression of other STAT family members.
    CONCLUSION: Our study presents a rich, multi-faceted summary of the molecular mechanisms impacted by STAT3 deletion and provides new insight for STAT3's potential as a therapeutic target in ovarian cancer.
    Keywords:  CRISPR-Cas9; Multi-omic genome-wide analysis; Ovarian cancer; STAT3; STAT3 knockout
  15. Nature. 2019 Sep 18.
      The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC)1,2. Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development2. Wild-type p53 is also known to modulate cellular metabolic pathways3, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of α-ketoglutarate (αKG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable αKG. Increased levels of the αKG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of αKG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase-an enzyme of the tricarboxylic acid cycle-specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of αKG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that αKG is an effector of p53-mediated tumour suppression, and that the accumulation of αKG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression.
  16. Anal Chem. 2019 Sep 17.
      mzTab 2.0 for metabolomics (mzTab-M) is the most recent standard format developed in collaboration by the Proteomics and Metabolomics Standards Initiatives including contributions by the recently founded Lipidomics Standards Initiative. mzTab-M is a redesign of the original mzTab format which was geared towards reporting of proteomics results, and as such provided only limited support for metabolites. As a tab-delimited, spread-sheet like format, mzTab-M captures experimental metadata, summary information on small molecules across assays, MS features as a basis for quantitation, and evidence to support reporting of individual or feature group identifications. Here, we present the Java reference implementation for reading, writing and validating mzTab-M files. Furthermore, we provide a web-application for conveniently validating mzTab-M files by a graphical user interface, and a command line validator that accompanies the library. The jmzTab-M library, version 1.0.41 is available at and from Maven Central at under the terms of the open source Apache License 2.0. The web-application, as well as the Python and R implementations are available at The respective websites link to additional API documentation, as well as to usage examples.