bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2022–12–04
sixteen papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh
  1. Ferroptosis induction via targeting metabolic alterations in head and neck cancer.
  2. High-throughput proteomics of nanogram-scale samples with Zeno SWATH MS.
  3. Rapid LC-MS assay for targeted metabolite quantification by serial injection into isocratic gradients.
  4. Critical assessment of chromatographic metadata in publicly available metabolomics data repositories.
  5. Comprehensive Online Reversed-Phase × Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid Analysis.
  6. Modified lipidomic profile of cancer-associated small extracellular vesicles facilitates tumorigenic behaviours and contributes to disease progression.
  7. Targeted profiling of polar metabolites in cancer metabolic reprogramming by hydrophilic interaction liquid chromatography-tandem mass spectrometry.
  8. Spatially resolved proteomics via tissue expansion.
  9. The octadecanoids: an emerging class of lipid mediators.
  10. A Mass Spectrometry-Based Strategy for Mapping Modification Sites for the Ubiquitin-Like Modifier NEDD8.
  11. Serum Metabolomics of Benign Essential Blepharospasm Using Liquid Chromatography and Orbitrap Mass Spectrometry.
  12. Protein-Peptide Turnover Profiling reveals the order of PTM addition and removal during protein maturation.
  13. Proteomic and functional profiling of platelet-derived extracellular vesicles released under physiological or tumor-associated conditions.
  14. Isotope tracing reveals distinct substrate preference in murine melanoma subtypes with differing anti-tumor immunity.
  15. Metabolon formation regulates branched-chain amino acid oxidation and homeostasis.
  16. Simultaneous quantification of tryptophan metabolites by liquid chromatography tandem mass spectrometry during early human pregnancy.
  1. Crit Rev Oncol Hematol. 2022 Nov 25. pii: S1040-8428(22)00311-0. [Epub ahead of print] 103887
    Ferroptosis induction via targeting metabolic alterations in head and neck cancer.
    Jaewang Lee, Jong-Lyel Roh.
      Ferroptosis is a newly regulated cell death induced by the accumulation of iron-mediated lipid peroxidation. The alteration of cancer metabolism may contribute to proliferation, metastasis, and treatment resistance in human cancers, implicating the sensitivity to ferroptosis induction. Altered metabolism in cancer cells regulates oxidative stresses and changes metabolism intermediates, contributing to their deregulated growth and proliferation. Cancer metabolic changes toward the elevation of cellular free iron and polyunsaturated fatty acids sensitize cancer cells to lipid peroxidation toxicity tightly linked to ferroptosis. The altered metabolism in cancers can be served as a promising target to reverse cancer therapeutic resistance by ferroptosis induction to selectively kill cancer cells while sparing normal cells. The role of mitochondria and lipid metabolism in inducing ferroptosis in head and neck cancer (HNC) has been elucidated in previous studies. Ferroptosis is receiving attention in cancer research as treating cancers altering cellular metabolism and refractory from conventional therapies. More in-depth studies are needed to develop highly therapeutic drugs and practical methods to induce ferroptosis in diverse cancer cells and tumor microenvironments effectively. Therefore, this review intends to understand the altered metabolism and find new therapeutic possibilities using ferroptosis in HNC.
    Keywords:  Metabolism; ferroptosis; head and neck cancer; lipid peroxidation; mitochondria
    DOI:  https://doi.org/10.1016/j.critrevonc.2022.103887
  2. Elife. 2022 Nov 30. pii: e83947. [Epub ahead of print]11
    High-throughput proteomics of nanogram-scale samples with Zeno SWATH MS.
    Ziyue Wang, Michael Mülleder, Ihor Batruch, Anjali Chelur, Kathrin Textoris-Taube, Torsten Schwecke, Johannes Hartl, Jason Causon, Jose Castro-Perez, Vadim Demichev, Stephen Tate, Markus Ralser.
      The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.
    Keywords:  biochemistry; chemical biology; data-independent acquisition; high-throughput screening; liquid chromatography; proteomics
    DOI:  https://doi.org/10.7554/eLife.83947
  3. Anal Bioanal Chem. 2022 Nov 28.
    Rapid LC-MS assay for targeted metabolite quantification by serial injection into isocratic gradients.
    Ryan A Groves, Carly C Y Chan, Spencer D Wildman, Daniel B Gregson, Thomas Rydzak, Ian A Lewis.
      Liquid chromatography mass spectrometry (LC-MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC-MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC-MS is throughput. Clinical LC-MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which-when analyzing complex metabolomics samples-are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC-MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC-MS tool for quantifying small sets of target biomarkers across large cohorts.
    Keywords:  Diagnostics; High-throughput screening; LC–MS; Metabolomics
    DOI:  https://doi.org/10.1007/s00216-022-04384-x
  4. Metabolomics. 2022 Nov 27. 18(12): 97
    Critical assessment of chromatographic metadata in publicly available metabolomics data repositories.
    Eva-Maria Harrieder, Fleming Kretschmer, Warwick Dunn, Sebastian Böcker, Michael Witting.
       INTRODUCTION: The structural identification of metabolites represents one of the current bottlenecks in non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics. The Metabolomics Standard Initiative has developed a multilevel system to report confidence in metabolite identification, which involves the use of MS, MS/MS and orthogonal data. Limitations due to similar or same fragmentation pattern (e.g. isomeric compounds) can be overcome by the additional orthogonal information of the retention time (RT), since it is a system property that is different for each chromatographic setup.
    OBJECTIVES: In contrast to MS data, sharing of RT data is not as widespread. The quality of data and its (re-)useability depend very much on the quality of the metadata. We aimed to evaluate the coverage and quality of this metadata from public metabolomics repositories.
    METHODS: We acquired an overview on the current reporting of chromatographic separation conditions. For this purpose, we defined the following information as important details that have to be provided: column name and dimension, flow rate, temperature, composition of eluents and gradient.
    RESULTS: We found that 70% of descriptions of the chromatographic setups are incomplete (according to our definition) and an additional 10% of the descriptions contained ambiguous and/or incorrect information. Accordingly, only about 20% of the descriptions allow further (re-)use of the data, e.g. for RT prediction. Therefore, we have started to develop a unified and standardized notation for chromatographic metadata with detailed and specific description of eluents, columns and gradients.
    CONCLUSION: Reporting of chromatographic metadata is currently not unified. Our recommended suggestions for metadata reporting will enable more standardization and automatization in future reporting.
    Keywords:  Data reuse; LC-MS; Metabolomics; Repositories; Retention time
    DOI:  https://doi.org/10.1007/s11306-022-01956-x
  5. Anal Chem. 2022 Nov 28.
    Comprehensive Online Reversed-Phase × Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid Analysis.
    Ryan Karongo, Jeannie Horak, Michael Lämmerhofer.
      This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral × chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC (1D), chiral recognition, and thus enantioselectivity by the core-shell tandem column composed of a quinine carbamate weak anion exchanger (QN-AX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) (2D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS2. The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core-shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC × LC separation occurs within a total run time of 60 min (1D), with the chiral 2D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-[U-13C15N]-AA standard. The quantification performance of this LC × LC-MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.
    DOI:  https://doi.org/10.1021/acs.analchem.2c03042
  6. Adv Biol Regul. 2022 Nov 21. pii: S2212-4926(22)00075-6. [Epub ahead of print] 100935
    Modified lipidomic profile of cancer-associated small extracellular vesicles facilitates tumorigenic behaviours and contributes to disease progression.
    Jordan Fyfe, Pratibha Malhotra, Marco Falasca.
      Metabolic rewiring is a key feature of cancer cells, which involves the alteration of amino acids, glucose and lipids to support aggressive cancer phenotypes. Changes in lipid metabolism alter cancer growth characteristics, membrane integrity and signalling pathways. Small extracellular vesicles (sEVs) are membrane-bound vesicles secreted by cells into the extracellular environment, where they participate in cell-to-cell communication. Lipids are involved in the formation and cargo assortment of sEVs, resulting in their selective packaging in these vesicles. Further, sEVs participate in different aspects of cancer development, such as proliferation, migration and angiogenesis. Various lipidomic studies have indicated the enrichment of specific lipids in sEVs derived from tumour cells, which aid in their pathological functioning. This paper summarises how the modified lipid profile of sEVs contributes to carcinogenesis and disease progression.
    Keywords:  Cancer biomarkers; Cancer progression; Extracellular vesicles; Lipid metabolism; Lipidomic
    DOI:  https://doi.org/10.1016/j.jbior.2022.100935
  7. J Chromatogr A. 2022 Dec 20. pii: S0021-9673(22)00845-7. [Epub ahead of print]1686 463654
    Targeted profiling of polar metabolites in cancer metabolic reprogramming by hydrophilic interaction liquid chromatography-tandem mass spectrometry.
    Xue-Man Dong, Pu Wu, Long-Hui Cheng, Lan Shou, Heng Dong, Xiao-Yu Chen, Hao-Jin Gao, Jian-Xiang Chen, Fei Xiang, Qi Zhang, Da-Hong Zhang, Jian-Liang Zhou, Tian Xie.
      Metabolic reprogramming of cancer cells is a hallmark of cancer, in which the polar metabolites involving aerobic glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and glutaminolysis play a crucial role in the occurrence and development of cancer. Therefore, targeted analysis of the polar metabolites in these pathways is of great value for understanding cancers, finding diagnostic biomarkers, and identifying therapeutic targets. However, it is still challenging to directly determine polar metabolites in these pathways without derivatization due to their diverse chemical properties, isomers, and strong polarity. Herein, a highly selective and sensitive HILIC-MS/MS method was developed for direct determination of the polar metabolites in aerobic glycolysis, PPP, TCA cycle, and glutaminolysis pathways. Without derivatization, 19 polar metabolites and their isomers with carbonyl, carboxyl, or phosphoryl groups in human plasma and cell extracts of prostate cancer (PC) were determined with strong retention and high resolution. This method has been widely verified by measuring linearity, precision, sensitivity, repeatability, matrix effect, and accuracy. The analysis of plasma samples by HILIC-MS/MS revealed distinct PC-specific metabolic signatures compared to a healthy control. In addition, this method could also be used to screen the targets of metabolic inhibitors at the cellular level. We conclude that the developed HILIC-MS/MS method provides a valuable means to study the cancer metabolic reprogramming or energy metabolism in living organisms.
    Keywords:  Cancer metabolic reprogramming; HILIC-MS/MS; Human plasma; Polar metabolites; Prostate cancer
    DOI:  https://doi.org/10.1016/j.chroma.2022.463654
  8. Nat Commun. 2022 Nov 30. 13(1): 7242
    Spatially resolved proteomics via tissue expansion.
    Lu Li, Cuiji Sun, Yaoting Sun, Zhen Dong, Runxin Wu, Xiaoting Sun, Hanbin Zhang, Wenhao Jiang, Yan Zhou, Xufeng Cen, Shang Cai, Hongguang Xia, Yi Zhu, Tiannan Guo, Kiryl D Piatkevich.
      Spatially resolved proteomics is an emerging approach for mapping proteome heterogeneity of biological samples, however, it remains technically challenging due to the complexity of the tissue microsampling techniques and mass spectrometry analysis of nanoscale specimen volumes. Here, we describe a spatially resolved proteomics method based on the combination of tissue expansion with mass spectrometry-based proteomics, which we call Expansion Proteomics (ProteomEx). ProteomEx enables quantitative profiling of the spatial variability of the proteome in mammalian tissues at ~160 µm lateral resolution, equivalent to the tissue volume of 0.61 nL, using manual microsampling without the need for custom or special equipment. We validated and demonstrated the utility of ProteomEx for streamlined large-scale proteomics profiling of biological tissues including brain, liver, and breast cancer. We further applied ProteomEx for identifying proteins associated with Alzheimer's disease in a mouse model by comparative proteomic analysis of brain subregions.
    DOI:  https://doi.org/10.1038/s41467-022-34824-2
  9. Biochem Soc Trans. 2022 Dec 01. pii: BST20210644. [Epub ahead of print]
    The octadecanoids: an emerging class of lipid mediators.
    Alessandro Quaranta, Johanna Revol-Cavalier, Craig E Wheelock.
      Oxylipins are enzymatic and non-enzymatic metabolites of mono- or polyunsaturated fatty acids that encompass potent lipid mediators including the eicosanoids and docosanoids. Previously considered of low interest and often dismissed as 'just fat', octadecanoid oxylipins have only recently begun to be recognized as lipid mediators in humans. In the last few years, these compounds have been found to be involved in the mediation of multiple biological processes related to nociception, tissue modulation, cell proliferation, metabolic regulation, inflammation, and immune regulation. At the same time, the study of octadecanoids is hampered by a lack of standardization in the field, a paucity of analytical standards, and a lack of domain expertise. These issues have collectively limited the investigation of the biosynthesis and bioactivity of octadecanoids. Here, we present an overview of the primary enzymatic pathways for the oxidative metabolism of 18-carbon fatty acids in humans and of the current knowledge of the major biological activity of the resulting octadecanoids. We also propose a systematic nomenclature system based upon that used for the eicosanoids in order to avoid ambiguities and resolve multiple designations for the same octadecanoid. The aim of this review is to provide an initial framework for the field and to assist in its standardization as well as to increase awareness of this class of compounds in order to stimulate research into this interesting group of lipid mediators.
    Keywords:  bioactive lipid; linoleic acid; lipid mediators; mass spectrometry; octadecanoid
    DOI:  https://doi.org/10.1042/BST20210644
  10. Methods Mol Biol. 2023 ;2602 137-149
    A Mass Spectrometry-Based Strategy for Mapping Modification Sites for the Ubiquitin-Like Modifier NEDD8.
    Caio A B Oliveira, Ekaterina Isaakova, Petra Beli, Dimitris P Xirodimas.
      The identification of modification sites for ubiquitin and ubiquitin-like modifiers is an essential step in the elucidation of controlled processes. The ubiquitin-like modifier NEDD8 is an important regulator of plethora of biological processes both under homeostatic and proteotoxic stress conditions. Here, we describe a detailed protocol for proteome-wide identification of NEDDylation sites. The approach is based on the use of cell lines stably expressing the NEDD8R74K mutant. Digestion of samples with Lysyl endopeptidase generates peptides with a di-glycine remnant only from proteins modified with NEDD8R74K but not with ubiquitin or ISG15. The isolation of these peptides with anti-di-glycine antibodies (K-ε-GG) allows the identification of NEDDylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS).
    Keywords:  Di-glycine remnant; High-throughput analysis; Mass spectrometry; NEDD8; Ubiquitin-like modifiers
    DOI:  https://doi.org/10.1007/978-1-0716-2859-1_10
  11. Oxid Med Cell Longev. 2022 ;2022 6876327
    Serum Metabolomics of Benign Essential Blepharospasm Using Liquid Chromatography and Orbitrap Mass Spectrometry.
    Han Zhao, Wushuang Wang, Tong Lin, Lan Gong.
       Background: Benign essential blepharospasm (BEB) is a form of focal dystonia that causes excessive involuntary spasms of the eyelids. Currently, the pathogenesis of BEB remains unclear. This study is aimed at investigating the serum metabolites profiles in patients with BEB and healthy control and to identify the mechanism and biomarkers of this disease.
    Methods: 30 patients with BEB and 33 healthy controls were recruited for this study. We conducted the quantitative and nontargeted metabolomics analysis of the serum samples from 63 subjects by using liquid chromatography and Orbitrap mass spectrometry (LC-Orbitrap MS). Multivariate statistical analysis was performed to detect and identify different metabolites between the two groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and receiver operating characteristic (ROC) curve analysis of the altered metabolites were performed.
    Results: A total of 134 metabolites were found and identified. The metabolites belonged to several metabolic pathways including phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis, linoleic acid metabolism, tryptophan metabolism, aminoacyl-tRNA biosynthesis, sphingolipid metabolism, glycosphingolipid biosynthesis, leucine and isoleucine biosynthesis, and vitamin B6 metabolism. Eight metabolites were identified as the potential biomarkers.
    Conclusions: These results demonstrated that serum metabolic profiling of BEB patients was significantly different from healthy controls based on LC-Orbitrap MS. Besides, metabolomics might provide useful information for a better understanding of BEB.
    DOI:  https://doi.org/10.1155/2022/6876327
  12. Nat Commun. 2022 Dec 02. 13(1): 7431
    Protein-Peptide Turnover Profiling reveals the order of PTM addition and removal during protein maturation.
    Henrik M Hammarén, Eva-Maria Geissen, Clement M Potel, Martin Beck, Mikhail M Savitski.
      Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods relying on pulsed metabolic labeling are popular to quantify turnover rates on a proteome-wide scale. Such data have traditionally been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a protein's lifetime-a fundamentally different metric. This is due to interconversion of the measured proteoform species. Using this framework, we identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results thus allow tying PTMs to the age of the modified proteins.
    DOI:  https://doi.org/10.1038/s41467-022-35054-2
  13. Cell Death Discov. 2022 Nov 26. 8(1): 467
    Proteomic and functional profiling of platelet-derived extracellular vesicles released under physiological or tumor-associated conditions.
    Mauro Vismara, Marcello Manfredi, Marta Zarà, Silvia Maria Grazia Trivigno, Luca Galgano, Silvia Stella Barbieri, Ilaria Canobbio, Mauro Torti, Gianni Francesco Guidetti.
      During hemostasis, thrombosis, and inflammation, activated blood platelets release extracellular vesicles (PEVs) that represent biological mediators of physiological and pathological processes. We have recently demonstrated that the activation of platelets by breast cancer cells is accompanied by a massive release of PEVs, evidence that matches with the observation that breast cancer patients display increased levels of circulating PEVs. A core concept in PEVs biology is that their nature, composition and biological function are strongly influenced by the conditions that induced their release. In this study we have performed a comparative characterization of PEVs released by platelets upon activation with thrombin, a potent thrombotic stimulus, and upon exposure to the breast cancer cell line MDA-MB-231. By nanoparticle tracking analysis and tandem mass spectrometry we have characterized the two populations of PEVs, showing that the thrombotic and tumoral stimuli produced vesicles that largely differ in protein composition. The bioinformatic analysis of the proteomic data led to the identification of signaling pathways that can be differently affected by the two PEVs population in target cells. Specifically, we have demonstrated that both thrombin- and cancer-cell-induced PEVs reduce the migration and potentiate Ca2+-induced apoptosis of Jurkat cells, but only thrombin-derived PEVs also potentiate cell necrosis. Our results demonstrate that stimulation of platelets by thrombotic or tumoral stimuli induces the release of PEVs with different protein composition that, in turn, may elicit selective biological responses in target cells.
    DOI:  https://doi.org/10.1038/s41420-022-01263-3
  14. Cancer Metab. 2022 Dec 01. 10(1): 21
    Isotope tracing reveals distinct substrate preference in murine melanoma subtypes with differing anti-tumor immunity.
    Xinyi Zhang, Alexandra A Halberstam, Wanling Zhu, Brooks P Leitner, Durga Thakral, Marcus W Bosenberg, Rachel J Perry.
       BACKGROUND: Research about tumor "metabolic flexibility"-the ability of cells to toggle between preferred nutrients depending on the metabolic context-has largely focused on obesity-associated cancers. However, increasing evidence for a key role for nutrient competition in the tumor microenvironment, as well as for substrate regulation of immune function, suggests that substrate metabolism deserves reconsideration in immunogenic tumors that are not strongly associated with obesity.
    METHODS: We compare two murine models: immunologically cold YUMM1.7 and immunologically-hot YUMMER1.7. We utilize stable isotope and radioisotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics analyses to comprehensively probe substrate preference in YUMM1.7 and YUMMER1.7 cells, with a subset of studies on the impact of available metabolites across a panel of five additional melanoma cell lines. We analyze bulk RNA-seq data and identify increased expression of amino acid and glucose metabolism genes in YUMMER1.7. Finally, we analyze melanoma patient RNA-seq data to identify potential prognostic predictors rooted in metabolism.
    RESULTS: We demonstrate using stable isotope tracer-based metabolic flux studies as well as gas and liquid chromatography-based metabolomics that immunologically-hot melanoma utilizes more glutamine than immunologically-cold melanoma in vivo and in vitro. Analyses of human melanoma RNA-seq data demonstrate that glutamine transporter and other anaplerotic gene expression positively correlates with lymphocyte infiltration and function.
    CONCLUSIONS: Here, we highlight the importance of understanding metabolism in non-obesity-associated cancers, such as melanoma. This work advances the understanding of the correlation between metabolism and immunogenicity in the tumor microenvironment and provides evidence supporting metabolic gene expression as potential prognostic factors of melanoma progression and may inform investigations of adjunctive metabolic therapy in melanoma.
    TRIAL REGISTRATION: Deidentified data from The Cancer Genome Atlas were analyzed.
    Keywords:  Amino acid; Glucose; Melanoma; Tumor metabolism; Tumor microenvironment
    DOI:  https://doi.org/10.1186/s40170-022-00296-7
  15. Nat Metab. 2022 Nov 28.
    Metabolon formation regulates branched-chain amino acid oxidation and homeostasis.
    McKenzie Patrick, Zhimin Gu, Gen Zhang, R Max Wynn, Pranita Kaphle, Hui Cao, Hieu Vu, Feng Cai, Xiaofei Gao, Yuannyu Zhang, Mingyi Chen, Min Ni, David T Chuang, Ralph J DeBerardinis, Jian Xu.
      The branched-chain aminotransferase isozymes BCAT1 and BCAT2, segregated into distinct subcellular compartments and tissues, initiate the catabolism of branched-chain amino acids (BCAAs). However, whether and how BCAT isozymes cooperate with downstream enzymes to control BCAA homeostasis in an intact organism remains largely unknown. Here, we analyse system-wide metabolomic changes in BCAT1- and BCAT2-deficient mouse models. Loss of BCAT2 but not BCAT1 leads to accumulation of BCAAs and branched-chain α-keto acids (BCKAs), causing morbidity and mortality that can be ameliorated by dietary BCAA restriction. Through proximity labelling, isotope tracing and enzymatic assays, we provide evidence for the formation of a mitochondrial BCAA metabolon involving BCAT2 and branched-chain α-keto acid dehydrogenase. Disabling the metabolon contributes to BCAT2 deficiency-induced phenotypes, which can be reversed by BCAT1-mediated BCKA reamination. These findings establish a role for metabolon formation in BCAA metabolism in vivo and suggest a new strategy to modulate this pathway in diseases involving dysfunctional BCAA metabolism.
    DOI:  https://doi.org/10.1038/s42255-022-00689-4
  16. Clin Chem Lab Med. 2022 Dec 05.
    Simultaneous quantification of tryptophan metabolites by liquid chromatography tandem mass spectrometry during early human pregnancy.
    Sofie K M van Zundert, Pieter H Griffioen, Lenie van Rossem, Sten P Willemsen, Yolanda B de Rijke, Ron H N van Schaik, Régine P M Steegers-Theunissen, Mina Mirzaian.
       OBJECTIVES: In this study we describe the development and validation of a liquid chromatography mass spectrometry method (LC-MS/MS) to quantify five tryptophan (TRP) metabolites within the kynurenine- and serotonin pathway and apply the method to serum samples of women in the first trimester of pregnancy. A secondary aim was to investigate the correlation between body mass index (BMI) and the five analytes.
    METHODS: A LC-MS/MS was developed for the analysis of TRP, kynurenine (KYN), 5-hydroxytryptophan (5-HTP), hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5-HIAA). Serum samples (n=374) were analyzed of pregnant women (median gestational age: 8 ± 2 weeks) participating in a subcohort of the Rotterdam Periconceptional Cohort (Predict study).
    RESULTS: The LC-MS/MS method provided satisfactory separation of the five analytes (7 min run). For all analytes R2 was >0.995. Within- and between-run accuracies were 72-97% and 79-104%, and the precisions were all <15% except for the between-run precisions of the low QC-samples of 5-HTP and 5-HT (both 16%). Analyte concentrations were determined in serum samples of pregnant women (median (IQR)); TRP (µmol/L): 57.5 (13.4), KYN (µmol/L): 1.4 (0.4), 5-HTP (nmol/L): 4.1 (1.2), 5-HT (nmol/L): 615 (323.1), and 5-HIAA (nmol/L): 39.9 (17.0). BMI was negatively correlated with TRP, 5-HTP, and 5-HIAA (TRP: r=-0.18, p<0.001; 5-HTP: r=-0.13, p=0.02; natural log of 5-HIAA: r=-0.11, p=0.04), and positively with KYN (r=0.11, p=0.04).
    CONCLUSIONS: The LC-MS/MS method is able to accurately quantify kynurenine- and serotonin pathway metabolites in pregnant women, providing an opportunity to investigate the role of the TRP metabolism in the (patho)physiology of pregnancy.
    Keywords:  liquid chromatography; mass spectrometry; pregnancy; tryptophan metabolism
    DOI:  https://doi.org/10.1515/cclm-2022-0790
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