bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒03‒14
thirty-two papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh
  1. Analysis of Melanoma Cell Glutamine Metabolism by Stable Isotope Tracing and Gas Chromatography-Mass Spectrometry.
  2. CD36-mediated ferroptosis dampens intratumoral CD8+ T cell effector function and impairs their antitumor ability.
  3. Metabolomics of sebum reveals lipid dysregulation in Parkinson's disease.
  4. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation.
  5. Loss of mitochondrial aconitase promotes colorectal cancer progression via SCD1-mediated lipid remodeling.
  6. Analyzing Melanoma Cell Oxygen Consumption and Extracellular Acidification Rates Using Seahorse Technology.
  7. Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides.
  8. Metabolite profiling of chondrosarcoma cells: A robust GC-MS method for the analysis of endogenous metabolome.
  9. Acetyl-CoA and Metabolite Fluxes Regulate White Adipose Tissue Expansion.
  10. SWATH-MS Protocols in Human Diseases.
  11. Rapid Shotgun Phosphoproteomics Analysis.
  12. Omega-3 fatty acids protect from colitis via an Alox15-derived eicosanoid.
  13. Polyporus Umbellatus Protects Against Renal Fibrosis by Regulating Intrarenal Fatty Acyl Metabolites.
  14. The glutamine antagonist prodrug JHU-083 slows malignant glioma growth and disrupts mTOR signaling.
  15. Qualitative and Quantitative Shotgun Proteomics Data Analysis from Data-Dependent Acquisition Mass Spectrometry.
  16. Quantification of Amine- and Alcohol-Containing Metabolites in Saline Samples Using Pre-extraction Benzoyl Chloride Derivatization and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).
  17. Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spec-trometry (GCIB-SIMS).
  18. Redox Epiphospholipidome in Programmed Cell Death Signaling: Catalytic Mechanisms and Regulation.
  19. High-Speed Screening of Lipoprotein Components Using Online Miniaturized Asymmetrical Flow Field-Flow Fractionation and Electrospray Ionization Tandem Mass Spectrometry: Application to Hepatocellular Carcinoma Plasma Samples.
  20. A SARS-CoV-2 Peptide Spectral Library Enables Rapid, Sensitive Identification of Virus Peptides in Complex Biological Samples.
  21. Ultra-High-Resolution IonStar Strategy Enhancing Accuracy and Precision of MS1-Based Proteomics and an Extensive Comparison with State-of-the-Art SWATH-MS in Large-Cohort Quantification.
  22. Systemic deletion of Atp7b modifies the hepatocytes' response to copper overload in the mouse models of Wilson disease.
  23. Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions.
  24. Postlymphadenectomy Analysis of Exosomes from Lymphatic Exudate/Exudative Seroma of Melanoma Patients.
  25. Clinical Proteomics for the Analysis of Circulating Extracellular Vesicles.
  26. Unbiased Microbiome and Metabolomic Profiling of Fecal Samples from Patients with Melanoma.
  27. Human Blood Plasma Investigation Employing 2D UPLC-UDMSE Data-Independent Acquisition Proteomics.
  28. The rawrr R Package: Direct Access to Orbitrap Data and Beyond.
  29. Metabolomic analyses of COVID-19 patients unravel stage-dependent and prognostic biomarkers.
  30. Binary Classifier for Computing Posterior Error Probabilities in MetaMorpheus.
  31. leapR: An R Package for Multiomic Pathway Analysis.
  32. PHONEMeS: Efficient Modeling of Signaling Networks Derived from Large-Scale Mass Spectrometry Data.
  1. Methods Mol Biol. 2021 ;2265 91-110
    Analysis of Melanoma Cell Glutamine Metabolism by Stable Isotope Tracing and Gas Chromatography-Mass Spectrometry.
    David A Scott.
      Glutamine is a major substrate for biosynthesis. It contributes to multiple pathways required for cell proliferation, supports antioxidant defense via glutathione synthesis, and sustains the tricarboxylic acid (TCA) cycle through anaplerosis. Glutamine-fueled anaplerosis and related biosynthesis can be studied in detail in melanoma using stable isotope (13C) labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis of metabolite amounts and labeling. Detailed protocols for the assay of polar metabolites (including amino acids, TCA cycle, and glycolysis metabolites) and fatty acids by these methods following cell treatment with 13C-glutamine or 13C-glucose are presented.
    Keywords:  Amino acids; Fatty acids; GC-MS; Glutamine; Glycolysis; Melanoma; Metabolite quantification; Stable-isotope tracing; Tricarboxylic acid cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1205-7_7
  2. Cell Metab. 2021 Mar 03. pii: S1550-4131(21)00071-1. [Epub ahead of print]
    CD36-mediated ferroptosis dampens intratumoral CD8+ T cell effector function and impairs their antitumor ability.
    Xingzhe Ma, Liuling Xiao, Lintao Liu, Lingqun Ye, Pan Su, Enguang Bi, Qiang Wang, Maojie Yang, Jianfei Qian, Qing Yi.
      Understanding the mechanisms underlying how T cells become dysfunctional in a tumor microenvironment (TME) will greatly benefit cancer immunotherapy. We found that increased CD36 expression in tumor-infiltrating CD8+ T cells, which was induced by TME cholesterol, was associated with tumor progression and poor survival in human and murine cancers. Genetic ablation of Cd36 in effector CD8+ T cells exhibited increased cytotoxic cytokine production and enhanced tumor eradication. CD36 mediated uptake of fatty acids by tumor-infiltrating CD8+ T cells in TME, induced lipid peroxidation and ferroptosis, and led to reduced cytotoxic cytokine production and impaired antitumor ability. Blocking CD36 or inhibiting ferroptosis in CD8+ T cells effectively restored their antitumor activity and, more importantly, possessed greater antitumor efficacy in combination with anti-PD-1 antibodies. This study reveals a new mechanism of CD36 regulating the function of CD8+ effector T cells and therapeutic potential of targeting CD36 or inhibiting ferroptosis to restore T cell function.
    Keywords:  CD36; CD8(+) T cells; ferroptosis; lipid peroxidation
    DOI:  https://doi.org/10.1016/j.cmet.2021.02.015
  3. Nat Commun. 2021 03 11. 12(1): 1592
    Metabolomics of sebum reveals lipid dysregulation in Parkinson's disease.
    Eleanor Sinclair, Drupad K Trivedi, Depanjan Sarkar, Caitlin Walton-Doyle, Joy Milne, Tilo Kunath, Anouk M Rijs, Rob M A de Bie, Royston Goodacre, Monty Silverdale, Perdita Barran.
      Parkinson's disease (PD) is a progressive neurodegenerative disorder, which is characterised by degeneration of distinct neuronal populations, including dopaminergic neurons of the substantia nigra. Here, we use a metabolomics profiling approach to identify changes to lipids in PD observed in sebum, a non-invasively available biofluid. We used liquid chromatography-mass spectrometry (LC-MS) to analyse 274 samples from participants (80 drug naïve PD, 138 medicated PD and 56 well matched control subjects) and detected metabolites that could predict PD phenotype. Pathway enrichment analysis shows alterations in lipid metabolism related to the carnitine shuttle, sphingolipid metabolism, arachidonic acid metabolism and fatty acid biosynthesis. This study shows sebum can be used to identify potential biomarkers for PD.
    DOI:  https://doi.org/10.1038/s41467-021-21669-4
  4. Nat Commun. 2021 03 11. 12(1): 1589
    mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation.
    Yilei Zhang, Robert V Swanda, Litong Nie, Xiaoguang Liu, Chao Wang, Hyemin Lee, Guang Lei, Chao Mao, Pranavi Koppula, Weijie Cheng, Jie Zhang, Zhenna Xiao, Li Zhuang, Bingliang Fang, Junjie Chen, Shu-Bing Qian, Boyi Gan.
      Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an essential role in inhibiting ferroptosis. As a selenoprotein, GPX4 protein synthesis is highly inefficient and energetically costly. How cells coordinate GPX4 synthesis with nutrient availability remains unclear. In this study, we perform integrated proteomic and functional analyses to reveal that SLC7A11-mediated cystine uptake promotes not only GSH synthesis, but also GPX4 protein synthesis. Mechanistically, we find that cyst(e)ine activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and promotes GPX4 protein synthesis at least partly through the Rag-mTORC1-4EBP signaling axis. We show that pharmacologic inhibition of mTORC1 decreases GPX4 protein levels, sensitizes cancer cells to ferroptosis, and synergizes with ferroptosis inducers to suppress patient-derived xenograft tumor growth in vivo. Together, our results reveal a regulatory mechanism to coordinate GPX4 protein synthesis with cyst(e)ine availability and suggest using combinatorial therapy of mTORC1 inhibitors and ferroptosis inducers in cancer treatment.
    DOI:  https://doi.org/10.1038/s41467-021-21841-w
  5. Mol Metab. 2021 Mar 03. pii: S2212-8778(21)00043-0. [Epub ahead of print] 101203
    Loss of mitochondrial aconitase promotes colorectal cancer progression via SCD1-mediated lipid remodeling.
    Xin You, Jingyu Tian, Hui Zhang, Yunhua Guo, Jing Yang, Chaofeng Zhu, Ming Song, Peng Wang, Zexian Liu, John Cancilla, Wenhua Lu, Christhophe Glorieux, Shijun Wen, Hongli Du, Peng Huang, Yumin Hu.
      OBJECTIVE: The mitochondrial aconitase (ACO2) is an essential enzyme that bridges TCA cycle and lipid metabolism. However, its role in cancer development remains to be elucidated. The metabolic subtype of colorectal cancer (CRC) has recently been established. We aim to investigate the potential role of ACO2 in CRC progression through mediating metabolic alterations.METHODS: We compared the mRNA and protein expression of ACO2 between paired CRC and non-tumor tissues from 353 patients. Correlation between ACO2 level and clinicopathological features was examined. CRC cell lines with knockdown or overexpression of ACO2 were analyzed for cell proliferation and tumor growth. Metabolomics and stable isotope tracing analysis were used to study the metabolic alterations induced by loss of ACO2.
    RESULTS: ACO2 was decreased in >50% of CRC samples compared with matched non-tumor tissues. Decreased ACO2 level was correlated with advanced disease stage (P<0.001) and shorter patient survival (P<0.001). Knockdown of ACO2 in CRC cells promoted cell proliferation and tumor formation, while ectopic expression of ACO2 restrained tumor growth. Specifically, blockade of ACO2 caused reduction in TCA cycle intermediates and suppression of mitochondrial oxidative phosphorylation, resulting in increase of glycolysis and elevated citrate flux for fatty acid and lipid synthesis. Increased citrate flux was able to induce upregulation of stearoyl-CoA desaturase (SCD1), which enhanced lipid desaturation in ACO2-deficent cells to favor colorectal cancer growth. Pharmacological inhibition of SCD selectively reduced tumor formation of CRC with ACO2 deficiency.
    CONCLUSIONS: Our study uncovers the rewiring metabolic pathway to maintain CRC survival during compromised TCA cycle and characterizes the therapeutic vulnerability of lipid desaturation in a meaningful subset of CRC with mitochondrial dysfunction.
    Keywords:  Colon cancer; Lipogenesis; Mitochondrial aconitase; Tricarboxylic acid (TCA) cycle; stearoyl-CoA desaturase
    DOI:  https://doi.org/10.1016/j.molmet.2021.101203
  6. Methods Mol Biol. 2021 ;2265 81-89
    Analyzing Melanoma Cell Oxygen Consumption and Extracellular Acidification Rates Using Seahorse Technology.
    Ashley V Menk, Greg M Delgoffe.
      Cancer cells have deregulated metabolism that can contribute to the unique metabolic makeup of the tumor microenvironment. This can be variable between patients, and it is important to understand these differences since they potentially can affect therapy response. Here we discuss a method of processing and assaying metabolism from direct ex vivo murine and human tumor samples using seahorse extracellular flux analysis. This provides real-time profiling of oxidative versus glycolytic metabolism and can help infer the metabolic status of the tumor microenvironment.
    Keywords:  Extracellular flux; Glycolysis; Metabolism; Oxidative phosphorylation; Seahorse
    DOI:  https://doi.org/10.1007/978-1-0716-1205-7_6
  7. Methods Mol Biol. 2021 ;2259 247-257
    Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides.
    Chia-Feng Tsai, Jeffrey S Smith, Dylan S Eiger, Kendall Martin, Tao Liu, Richard D Smith, Tujin Shi, Sudarshan Rajagopal, Jon M Jacobs.
      Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.
    Keywords:  Hydrophilic phosphopeptide; Mass spectrometry; Phosphopeptide enrichment; Phosphoproteomics; TMT labeling
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_16
  8. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Feb 27. pii: S1570-0232(21)00086-6. [Epub ahead of print]1169 122606
    Metabolite profiling of chondrosarcoma cells: A robust GC-MS method for the analysis of endogenous metabolome.
    Jinfeng Zhu, Mengmeng Zhang, Jinxiu Guo, Xueke Wu, Shaomin Wang, Yubing Zhou, Hongmin Liu.
      Chondrosarcoma is the 2nd most frequent bone sarcoma. In this study, the metabolic profiling of human chondrosarcoma SW-1353 cell line was investigated for the first time. To obtain more precise information about the metabolites from chondrosarcoma cells, pretreatment methods including washing steps/solvents, harvesting conditions, and extraction protocols for chondrosarcoma SW-1353 cells were evaluated in the context of metabolite profiling by GC-MS technique. In addition, a total of 32 representative metabolites (related to amino acid metabolism, TCA cycle, glycolysis, and fatty acid metabolism) were quantitatively determined. We found that a fast water rinse step before metabolic quenching, may reduce the contaminants and improve sensitivity. Trypsin/ethylene diamine tetraacetic acid treatment led to a large amount of metabolite leakage, which was not suitable for metabolomics research. Methanol was selected as a more suitable extraction solvent among four extraction approaches applied to SW-1353 cells. The final protocol can provide a simple, robust, and reproducible method to obtain precise information about the metabolites from chondrosarcoma cells, which is helpful to further understand the chondrosarcoma cell physiology and the mechanism of drug resistance in this disease, from the perspective of metabolomics.
    Keywords:  Chondrosarcoma cells; Gas chromatograph-mass spectrometry; Metabolomics; Sample pretreatment
    DOI:  https://doi.org/10.1016/j.jchromb.2021.122606
  9. Trends Endocrinol Metab. 2021 Mar 09. pii: S1043-2760(21)00043-6. [Epub ahead of print]
    Acetyl-CoA and Metabolite Fluxes Regulate White Adipose Tissue Expansion.
    Jessica B Felix, Aaron R Cox, Sean M Hartig.
      White adipose tissue (WAT) depends on coordinated regulation of transcriptional and metabolic pathways to respond to whole-body energy demands. We highlight metabolites that contribute to biosynthetic reactions for WAT expansion. Recent studies have precisely defined how byproducts of carbohydrate and lipid metabolism affect physiological and endocrine functions in adipocytes. We emphasize the critical emerging roles of short-chain fatty acids (SCFAs) and tricarboxylic acid (TCA) cycle metabolites that connect lipogenesis to WAT energy balance and endocrine functions. These insights address how adipocytes use small molecules generated from central carbon metabolism to measure responses to nutritional stress.
    Keywords:  adipose tissue; insulin; lipid metabolism; metabolite; microenvironment
    DOI:  https://doi.org/10.1016/j.tem.2021.02.008
  10. Methods Mol Biol. 2021 ;2259 105-141
    SWATH-MS Protocols in Human Diseases.
    Maria Del Pilar Chantada-Vázquez, María García Vence, Antonio Serna, Cristina Núñez, Susana B Bravo.
      Identification of molecular biomarkers for human diseases is one of the most important disciplines in translational science as it helps to elucidate their origin and early progression. Thus, it is a key factor in better diagnosis, prognosis, and treatment. Proteomics can help to solve the problem of sample complexity when the most common primary sample specimens were analyzed: organic fluids of easy access. The latest developments in high-throughput and label-free quantitative proteomics (SWATH-MS), together with more advanced liquid chromatography, have enabled the analysis of large sample sets with the sensitivity and depth needed to succeed in this task. In this chapter, we show different sample processing methods (major protein depletion, digestion, etc.) and a micro LC-SWATH-MS protocol to identify/quantify several proteins in different types of samples (serum/plasma, saliva, urine, tears).
    Keywords:  Biological fluids; Label-free quantification; Nanoproteomics; SWATH-MS; Saliva; Serum/plasma; Tears; Urine
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_7
  11. Methods Mol Biol. 2021 ;2259 259-268
    Rapid Shotgun Phosphoproteomics Analysis.
    Mónica Carrera, Benito Cañas, Daniel Lopez-Ferrer.
      In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by high-intensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO2) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer (LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat T-cells. Using this accelerated workflow, HIFU-TiO2-SCX-LC-MS/MS, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently identified in less than 15 h.
    Keywords:  High-intensity focused ultrasound (HIFU); Jurkat T-cells; Mass spectrometry (MS); Phosphoproteomics; Proteomics; Strong cation exchange chromatography (SCX); Titanium dioxide (TiO2)
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_17
  12. FASEB J. 2021 Apr;35(4): e21491
    Omega-3 fatty acids protect from colitis via an Alox15-derived eicosanoid.
    Nadine Rohwer, Cheng-Ying Chiu, Dan Huang, Christopher Smyl, Michael Rothe, Katharina M Rund, Nils Helge Schebb, Hartmut Kühn, Karsten-Henrich Weylandt.
      An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) tissue status can lead to a significant formation of anti-inflammatory lipid mediators and effective reduction in inflammation and tissue injury in murine colitis. Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory bowel disease as well as in the formation of pro- and anti-inflammatory lipid mediators. To explore the role of Alox15 in the protective response found in fat1 transgenic mice with endogenously increased n-3 PUFA tissue status fat1 transgenic mice were crossed with Alox15-deficient animals and challenged in the dextran sulfate sodium (DSS)- and the 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis model. Transgenic fat1 mice rich in endogenous n-3 PUFAs were protected from colitis. However, additional systemic inactivation of the Alox15 gene counteracted this protective effect. To explore the molecular basis for this effect Alox15 lipid metabolites derived from n-3 PUFA were analyzed in the different mice. Alox15 deficiency suppressed the formation of n-3 PUFA-derived 15-hydroxy eicosapentaenoic acid (15-HEPE). In contrast, treating mice with intraperitoneal injections of 15S-HEPE protected wild-type mice from DSS- and TNBS-induced colitis. These data suggest that the anti-colitis effect of increased n-3 PUFA in the transgenic fat1 mouse model is mediated in part via Alox15-derived 15-HEPE formation.
    Keywords:  eicosanoids; inflammatory bowel disease; lipid peroxidation; lipoxygenases
    DOI:  https://doi.org/10.1096/fj.202002340RR
  13. Front Pharmacol. 2021 ;12 633566
    Polyporus Umbellatus Protects Against Renal Fibrosis by Regulating Intrarenal Fatty Acyl Metabolites.
    Yan-Ni Wang, Xia-Qing Wu, Dan-Dan Zhang, He-He Hu, Jian-Ling Liu, Nosratola D Vaziri, Yan Guo, Ying-Yong Zhao, Hua Miao.
      Background: Chronic renal failure (CRF) results in significant dyslipidemia and profound changes in lipid metabolism. Polyporus umbellatus (PPU) has been shown to prevent kidney injury and subsequent kidney fibrosis. Methods: Lipidomic analysis was performed to explore the intrarenal profile of lipid metabolites and further investigate the effect of PPU and its main bioactive component, ergone, on disorders of lipid metabolism in rats induced by adenine. Univariate and multivariate statistical analyses were performed for choosing intrarenal differential lipid species in CRF rats and the intervening effect of n-hexane extract of PPU and ergone on CRF rats. Results: Compared with control group, decreased creatinine clearance rate indicated declining kidney function in CRF group. Based on the lipidomics, we identified 65 lipid species that showed significant differences between CRF and control groups. The levels of 12 lipid species, especially fatty acyl lipids including docosahexaenoic acid, docosapentaenoic acid (22n-3), 10,11-Dihydro-12R-hydroxy-leukotriene C4, 3-hydroxydodecanoyl carnitine, eicosapentaenoic acid, hypogeic acid and 3-hydroxypentadecanoic acid had a strong linear correlation with creatinine clearance rate, which indicated these lipid species were associated with impaired renal function. In addition, receiver operating characteristics analysis showed that 12 lipid species had high area under the curve values with high sensitivity and specificity for differentiating CRF group from control group. These changes are related to the perturbation of fatty acyl metabolism. Treatment with PPU and ergone improved the impaired kidney function and mitigated renal fibrosis. Both chemometrics and cluster analyses showed that rats treated by PPU and ergone could be separated from CRF rats by using 12 lipid species. Intriguingly, PPU treatment could restore the levels of 12 lipid species, while treatment with ergone could only reverse the changes of six fatty acids in CRF rats. Conclusion: Altered intrarenal fatty acyl metabolites were implicated in pathogenesis of renal fibrosis. PPU and ergone administration alleviated renal fibrosis and partially improved fatty acyl metabolism. These findings suggest that PPU exerted its renoprotective effect by regulating fatty acyl metabolism as a potential biochemical mechanism. Therefore, these findings indicated that fatty acyl metabolism played an important role in renal fibrosis and could be considered as an effective therapeutic avenue against renal fibrosis.
    Keywords:  Polyporus umbellatus; chronic renal failure; ergone; fatty acid metabolism; lipidomics; mass spectrometry; ultra-performance liquid chromatography
    DOI:  https://doi.org/10.3389/fphar.2021.633566
  14. Neurooncol Adv. 2021 Jan-Dec;3(1):3(1): vdaa149
    The glutamine antagonist prodrug JHU-083 slows malignant glioma growth and disrupts mTOR signaling.
    Alex Shimura Yamashita, Marina da Costa Rosa, Vittorio Stumpo, Rana Rais, Barbara S Slusher, Gregory J Riggins.
      Background: Metabolic reprogramming is a common feature in cancer, and it is critical to facilitate cancer cell growth. Isocitrate Dehydrogenase 1/2 (IDH1 and IDH2) mutations (IDHmut) are the most common genetic alteration in glioma grade II and III and secondary glioblastoma and these mutations increase reliance on glutamine metabolism, suggesting a potential vulnerability. In this study, we tested the hypothesis that the brain penetrant glutamine antagonist prodrug JHU-083 reduces glioma cell growth.Material and Methods: We performed cell growth, cell cycle, and protein expression in glutamine deprived or Glutaminase (GLS) gene silenced glioma cells. We tested the effect of JHU-083 on cell proliferation, metabolism, and mTOR signaling in cancer cell lines. An orthotopic IDH1R132H glioma model was used to test the efficacy of JHU-083 in vivo.
    Results: Glutamine deprivation and GLS gene silencing reduced glioma cell proliferation in vitro in glioma cells. JHU-083 reduced glioma cell growth in vitro, modulated cell metabolism, and disrupted mTOR signaling and downregulated Cyclin D1 protein expression, through a mechanism independent of TSC2 modulation and glutaminolysis. IDH1R132H isogenic cells preferentially reduced cell growth and mTOR signaling downregulation. In addition, guanine supplementation partially rescued IDHmut glioma cell growth, mTOR signaling, and Cyclin D1 protein expression in vitro. Finally, JHU-083 extended survival in an intracranial IDH1 mut glioma model and reduced intracranial pS6 protein expression.
    Conclusion: Targeting glutamine metabolism with JHU-083 showed efficacy in preclinical models of IDHmut glioma and measurably decreased mTOR signaling.
    Keywords:  IDH mutation; cell cycle; glioma; glutamine metabolism; mTOR signaling
    DOI:  https://doi.org/10.1093/noajnl/vdaa149
  15. Methods Mol Biol. 2021 ;2259 297-308
    Qualitative and Quantitative Shotgun Proteomics Data Analysis from Data-Dependent Acquisition Mass Spectrometry.
    Jesse G Meyer.
      Shotgun proteomics is the inferential analysis of proteoforms using peptide proxies produced by enzyme-catalyzed hydrolysis of entire proteomes. Such peptides are usually identified by nanoflow liquid chromatography coupled to tandem mass spectrometry analysis (nLC-MS/MS). Traditionally, MS/MS analysis is performed in data-dependent acquisition (DDA) mode, which usually produces a pattern of fragment masses unique to a single peptide's fragmentation. Here, I describe a statistically rigorous qualitative and quantitative computational analysis for shotgun proteomics DDA analysis using free open-source software tools. MS/MS data are used to identify peptides, and the area of peptide mass/charge over chromatographic elution is used to quantify peptides. All peptides that uniquely map to a protein sequence predicted from the genome are combined into a single protein quantity, which can then be compared across experimental conditions. Statistically significant protein changes can be summarized using gene ontology or pathway term enrichment analysis.
    Keywords:  Data-dependent acquisition; Gene ontology analysis; Mass spectrometry; Pathway analysis; Peptides; Proteomics; Quantification
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_19
  16. Anal Chem. 2021 Mar 10.
    Quantification of Amine- and Alcohol-Containing Metabolites in Saline Samples Using Pre-extraction Benzoyl Chloride Derivatization and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).
    Brittany Widner, Melissa C Kido Soule, Frank Xavier Ferrer-González, Mary Ann Moran, Elizabeth B Kujawinski.
      Dissolved metabolites serve as nutrition, energy, and chemical signals for microbial systems. However, the full scope and magnitude of these processes in marine systems are unknown, largely due to insufficient methods, including poor extraction of small, polar compounds using common solid-phase extraction resins. Here, we utilized pre-extraction derivatization and ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to detect and quantify targeted dissolved metabolites in seawater and saline culture media. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-ISs) produced from 13C6-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples and evaluated matrix-derived biases. We have optimized this quantitative method for 73 common metabolites, of which 50 cannot be quantified without derivatization due to low extraction efficiencies. Of the 73 metabolites, 66 were identified in either culture media or seawater and 45 of those were quantified. This derivatization method is sensitive (detection limits = pM to nM), rapid (∼5 min per sample), and high throughput.
    DOI:  https://doi.org/10.1021/acs.analchem.0c03769
  17. Angew Chem Int Ed Engl. 2021 Mar 08.
    Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spec-trometry (GCIB-SIMS).
    Louis Sparvero, Hua Tian, Andrew A Amoscato, Wan-Yang Sun, Tamil S Anthonymuthu, Yulia Y Tyurina, Oleksandr Kapralov, Sabzali Javadov, Rong-Rong He, Simon C Watkins, Nicholas Winograd, Valerian E Kagan, Hülya Bayır.
      Peroxidized phosphatidylethanolamine (PEox) species have been identified by Liquid Chromatography Mass Spectrometry (LC-MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying Gas Cluster Ion Beam Secondary Ion Mass Spectrometry (GCIB-SIMS) imaging with a 70 keV (H 2 O) n + (n>28,000) cluster ion beam, we were able to map PEox with 1.2 micron spatial resolution at the single cell/subcellular level in ferroptotic H9c2 cardiomyocytes and cortical/hippocampal neurons after traumatic brain injury. Application of this protocol affords visualization of physiologically relevant levels of very low abundance (20 pmol/μmol lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.
    Keywords:  Cell Death; Lipid; imaging; mass spectrometry; oxidation
    DOI:  https://doi.org/10.1002/anie.202102001
  18. Front Endocrinol (Lausanne). 2020 ;11 628079
    Redox Epiphospholipidome in Programmed Cell Death Signaling: Catalytic Mechanisms and Regulation.
    Valerian E Kagan, Yulia Y Tyurina, Irina I Vlasova, Alexander A Kapralov, Andrew A Amoscato, Tamil S Anthonymuthu, Vladimir A Tyurin, Indira H Shrivastava, Fatma B Cinemre, Andrew Lamade, Michael W Epperly, Joel S Greenberger, Donald H Beezhold, Rama K Mallampalli, Apurva K Srivastava, Hulya Bayir, Anna A Shvedova.
      A huge diversification of phospholipids, forming the aqueous interfaces of all biomembranes, cannot be accommodated within a simple concept of their role as membrane building blocks. Indeed, a number of signaling functions of (phospho)lipid molecules has been discovered. Among these signaling lipids, a particular group of oxygenated polyunsaturated fatty acids (PUFA), so called lipid mediators, has been thoroughly investigated over several decades. This group includes oxygenated octadecanoids, eicosanoids, and docosanoids and includes several hundreds of individual species. Oxygenation of PUFA can occur when they are esterified into major classes of phospholipids. Initially, these events have been associated with non-specific oxidative injury of biomembranes. An alternative concept is that these post-synthetically oxidatively modified phospholipids and their adducts with proteins are a part of a redox epiphospholipidome that represents a rich and versatile language for intra- and inter-cellular communications. The redox epiphospholipidome may include hundreds of thousands of individual molecular species acting as meaningful biological signals. This review describes the signaling role of oxygenated phospholipids in programs of regulated cell death. Although phospholipid peroxidation has been associated with almost all known cell death programs, we chose to discuss enzymatic pathways activated during apoptosis and ferroptosis and leading to peroxidation of two phospholipid classes, cardiolipins (CLs) and phosphatidylethanolamines (PEs). This is based on the available LC-MS identification and quantitative information on the respective peroxidation products of CLs and PEs. We focused on molecular mechanisms through which two proteins, a mitochondrial hemoprotein cytochrome c (cyt c), and non-heme Fe lipoxygenase (LOX), change their catalytic properties to fulfill new functions of generating oxygenated CL and PE species. Given the high selectivity and specificity of CL and PE peroxidation we argue that enzymatic reactions catalyzed by cyt c/CL complexes and 15-lipoxygenase/phosphatidylethanolamine binding protein 1 (15LOX/PEBP1) complexes dominate, at least during the initiation stage of peroxidation, in apoptosis and ferroptosis. We contrast cell-autonomous nature of CLox signaling in apoptosis correlating with its anti-inflammatory functions vs. non-cell-autonomous ferroptotic signaling facilitating pro-inflammatory (necro-inflammatory) responses. Finally, we propose that small molecule mechanism-based regulators of enzymatic phospholipid peroxidation may lead to highly specific anti-apoptotic and anti-ferroptotic therapeutic modalities.
    Keywords:  apoptosis; cardiolipin; cytochrome c; ferroptosis; lipoxygenase; phospholipid peroxidation; redox lipidomics; regulated cell death
    DOI:  https://doi.org/10.3389/fendo.2020.628079
  19. Anal Chem. 2021 Mar 10.
    High-Speed Screening of Lipoprotein Components Using Online Miniaturized Asymmetrical Flow Field-Flow Fractionation and Electrospray Ionization Tandem Mass Spectrometry: Application to Hepatocellular Carcinoma Plasma Samples.
    Jin Yong Kim, Gwang Bin Lee, Jong Cheol Lee, Myeong Hee Moon.
      This study introduces a high-speed screening method for the quantitative analysis of lipoprotein components in human plasma samples using online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-tandem mass spectrometry (mAF4-ESI-MS/MS). Using an mAF4 channel, high-density lipoproteins and low-density lipoproteins can be fractionated by size at a high speed (<10 min) and directly fed to ESI-MS/MS for the simultaneous screening of targeted lipid species and apolipoprotein A1 (ApoA1). By employing the heated electrospray ionization probe as an ionization source, an mAF4 effluent flow rate of up to a few tens of microliters per minute can be used, which is adequate for direct feeding to MS without splitting the outflow, resulting in a consistent feed rate to MS for stable MS detection. mAF4-ESI-MS/MS was applied to hepatocellular carcinoma (HCC) plasma samples for targeted quantification of 25 lipid biomarker candidates and ApoA1 compared with healthy controls, the results of which were in statistical agreement with the quantified results obtained by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry. Moreover, the present method provided the simultaneous detection of changes in lipoprotein size and the relative amount. This study demonstrated the potential of mAF4-ESI-MS/MS as an alternative high-speed screening platform for the top-down analysis of targeted lipoprotein components in patients with HCC, which is applicable to other diseases that involve the perturbation of lipoproteins.
    DOI:  https://doi.org/10.1021/acs.analchem.0c04756
  20. J Proteome Res. 2021 Mar 08.
    A SARS-CoV-2 Peptide Spectral Library Enables Rapid, Sensitive Identification of Virus Peptides in Complex Biological Samples.
    Jonathan R St-Germain, Audrey Astori, Brian Raught.
      On the basis of an analysis of (i) SARS-CoV-2 virions, (ii) SARS-CoV-2-infected VeroE6 cell lysates, and (iii) recombinant SARS-CoV-2 proteins expressed in HEK 293 cells, here we present a comprehensive SARS-CoV-2 peptide spectrum compendium, comprising 1682 high confidence peptide consensus spectra derived from 1170 peptides (of various charge states) spanning 23 virus proteins. This high quality reference set can be used, e.g., for the selection of commonly observed virus peptides for use in targeted proteomics or data-independent acquisition (DIA) approaches. Using this rich resource, we also demonstrate that a spectral matching search approach yields improved performance over the use of standard database search engines alone for the identification of virus peptides in complex biological samples.
    Keywords:  COVID-19; SARS-CoV-2; SpectraST; coronavirus; mass spectrometry; proteomics; spectral library; spectral matching
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00048
  21. Anal Chem. 2021 Mar 09.
    Ultra-High-Resolution IonStar Strategy Enhancing Accuracy and Precision of MS1-Based Proteomics and an Extensive Comparison with State-of-the-Art SWATH-MS in Large-Cohort Quantification.
    Xue Wang, Liang Jin, Chenqi Hu, Shichen Shen, Shuo Qian, Min Ma, Xiaoyu Zhu, Fengzhi Li, Jianmin Wang, Yu Tian, Jun Qu.
      Quantitative proteomics in large cohorts is highly valuable for clinical/pharmaceutical investigations but often suffers from severely compromised reliability, accuracy, and reproducibility. Here, we describe an ultra-high-resolution IonStar method achieving reproducible protein measurement in large cohorts while minimizing the ratio compression problem, by taking advantage of the exceptional selectivity of ultra-high-resolution (UHR)-MS1 detection (240k_FWHM@m/z = 200). Using mixed-proteome benchmark sets reflecting large-cohort analysis with technical or biological replicates (N = 56), we comprehensively compared the quantitative performances of UHR-IonStar vs a state-of-the-art SWATH-MS method, each with their own optimal analytical platforms. We confirmed a cutting-edge micro-liquid chromatography (LC)/Triple-TOF with Spectronaut outperforms nano-LC/Orbitrap for SWATH-MS, which was then meticulously developed/optimized to maximize sensitivity, reproducibility, and proteome coverage. While the two methods with distinct principles (i.e., MS1- vs MS2-based) showed similar depth-of-analysis (∼6700-7000 missing-data-free proteins quantified, 1% protein-false discovery rate (FDR) for entire set, 2 unique peptides/protein) and good accuracy/precision in quantifying high-abundance proteins, UHR-IonStar achieved substantially superior quantitative accuracy, precision, and reproducibility for lower-abundance proteins (a category that includes most regulatory proteins), as well as much-improved sensitivity/selectivity for discovering significantly altered proteins. Furthermore, compared to SWATH-MS, UHR-IonStar showed markedly higher accuracy for a single analysis of each sample across a large set, which is an inadequately investigated albeit critical parameter for large-cohort analysis. Finally, we compared UHR-IonStar vs SWATH-MS in measuring the time courses of altered proteins in paclitaxel-treated cells (N = 36), where dysregulated biological pathways have been very well established. UHR-IonStar discovered substantially more well-recognized biological processes/pathways induced by paclitaxel. Additionally, UHR-IonStar showed markedly superior ability than SWATH-MS in accurately depicting the time courses of well known to be paclitaxel-induced biomarkers. In summary, UHR-IonStar represents a reliable, robust, and cost-effective solution for large-cohort proteomic quantification with excellent accuracy and precision.
    DOI:  https://doi.org/10.1021/acs.analchem.0c05002
  22. Sci Rep. 2021 Mar 11. 11(1): 5659
    Systemic deletion of Atp7b modifies the hepatocytes' response to copper overload in the mouse models of Wilson disease.
    Abigael Muchenditsi, C Conover Talbot, Aline Gottlieb, Haojun Yang, Byunghak Kang, Tatiana Boronina, Robert Cole, Li Wang, Som Dev, James P Hamilton, Svetlana Lutsenko.
      Wilson disease (WD) is caused by inactivation of the copper transporter Atp7b and copper overload in tissues. Mice with Atp7b deleted either globally (systemic inactivation) or only in hepatocyte recapitulate various aspects of human disease. However, their phenotypes vary, and neither the common response to copper overload nor factors contributing to variability are well defined. Using metabolic, histologic, and proteome analyses in three Atp7b-deficient mouse strains, we show that global inactivation of Atp7b enhances and specifically modifies the hepatocyte response to Cu overload. The loss of Atp7b only in hepatocytes dysregulates lipid and nucleic acid metabolisms and increases the abundance of respiratory chain components and redox balancing enzymes. In global knockouts, independently of their background, the metabolism of lipid, nucleic acid, and amino acids is inhibited, respiratory chain components are down-regulated, inflammatory response and regulation of chromosomal replication are enhanced. Decrease in glucokinase and lathosterol oxidase and elevation of mucin-13 and S100A10 are observed in all Atp7b mutant strains and reflect the extent of liver injury. The magnitude of proteomic changes in Atp7b-/- animals inversely correlates with the metallothioneins levels rather than liver Cu content. These findings facilitate identification of WD-specific metabolic and proteomic changes for diagnostic and treatment.
    DOI:  https://doi.org/10.1038/s41598-021-84894-3
  23. Methods Mol Biol. 2021 ;2259 25-45
    Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions.
    Ana Gordon, Karine Gousset.
      Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions-such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.-are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enable the protein profiling of these small and diverse subcellular structures.
    Keywords:  Cellular protrusion; DTBP; Fixation; Laser capture microdissection; Mass spectrometry; Proteomics
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_3
  24. Methods Mol Biol. 2021 ;2265 345-359
    Postlymphadenectomy Analysis of Exosomes from Lymphatic Exudate/Exudative Seroma of Melanoma Patients.
    Susana García-Silva, Pilar Ximénez-Embún, Javier Muñoz, Héctor Peinado.
      Circulating extracellular vesicles in biofluids have become an interesting approach to analyse disease biomarkers. There are multiple methods for isolation of extracellular vesicles, though differential ultracentrifugation is still considered as the gold-standard isolation technique for exosomes. Furthermore, exosomes purified by this method have been demonstrated to display functional activity in vitro and in vivo and exhibit great versatility for subsequent analysis including proteomics, electron microscopy, mass spectrometry, or nucleic acid analysis. Here, we describe the method for isolation of exosomes from lymphatic exudate (seroma) obtained postlymphadenectomy for liquid biopsy approaches.
    Keywords:  Exosomes; Extracellular vesicles; Iodixanol gradient; Lymphadenectomy; Lymphatic exudate; Ultracentrifugation
    DOI:  https://doi.org/10.1007/978-1-0716-1205-7_25
  25. Methods Mol Biol. 2021 ;2259 13-23
    Clinical Proteomics for the Analysis of Circulating Extracellular Vesicles.
    Maria N Barrachina, Ángel García.
      In recent years, technical improvements in proteomics have allowed its rapid application for biomarker discovery, new drug target identification, and the study of disease progression and drug resistance. The clinical potential of circulating extracellular vesicles (EVs) as a source of biomarkers is one of the reasons why several research groups have recently applied proteomics to their study. A large variety of proteomic approaches such as gel-based proteomics and bottom-up and top-down mass spectrometry have been applied to the study of EVs. In this chapter, we will present basic protocols for gel-based and quantitative MS-based approaches applied to the study of EVs.
    Keywords:  2D-DIGE; Extracellular vesicles; Label-free LC-MS/MS; Platelets
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_2
  26. Methods Mol Biol. 2021 ;2265 461-474
    Unbiased Microbiome and Metabolomic Profiling of Fecal Samples from Patients with Melanoma.
    Ashley Bui, Yongbin Choi, Arthur E Frankel, Andrew Y Koh.
      Gut microbiota influence and modulate host immune responses. In preclinical cancer models, mice lacking gut microbiota have a markedly diminished response to immune checkpoint inhibitor therapy. Further, in melanoma patients, specific commensal gut microbiota have been associated with a positive clinical response to immunotherapy. In order to study the gut microbiome and metabolome, we have developed methods for fecal sample collection and processing, microbiome and metabolome profiling, and bioinformatic analysis. This protocol will be a useful tool for interrogating the taxonomic composition and functional output of a melanoma patient's gut microbiome.
    Keywords:  Fecal; Immunotherapy; Melanoma; Metabolomics; Microbiome
    DOI:  https://doi.org/10.1007/978-1-0716-1205-7_33
  27. Methods Mol Biol. 2021 ;2259 153-165
    Human Blood Plasma Investigation Employing 2D UPLC-UDMSE Data-Independent Acquisition Proteomics.
    Licia C Silva-Costa, Bradley J Smith, Pamela T Carlson, Gustavo H M F Souza, Daniel Martins-de-Souza.
      Proteomic tools are especially useful when it comes to investigating complex samples such as human blood plasma, in which protein quantities can span across up to ten orders of magnitude. Ultra definition mass spectrometry, in combination with two-dimensional liquid chromatography, provides better coverage of complex proteomes and allows for better control of collision energy, keeping the fragmentation benefits of high collision energy associated with drift time measurements from ion mobility separation. Here, we present a protocol to assist in the identification of proteins in human blood plasma and other similar samples with a large dynamic range.
    Keywords:  Depletion; Immunoaffinity depletion; Mass spectrometry; Multifactorial disorders; Serum; Tryptic digestion
    DOI:  https://doi.org/10.1007/978-1-0716-1178-4_9
  28. J Proteome Res. 2021 Mar 09.
    The rawrr R Package: Direct Access to Orbitrap Data and Beyond.
    Tobias Kockmann, Christian Panse.
      The Bioconductor project (Nat. Methods 2015, 12 (2), 115-121) has shown that the R statistical environment is a highly valuable tool for genomics data analysis, but with respect to proteomics, we are still missing low-level infrastructure to enable performant and robust analysis workflows in R. Fundamentally important are libraries that provide raw data access. Our R package rawDiag (J. Proteome Res. 2018, 17 (8), 2908-2914) has provided the proof-of-principle how access to mass spectrometry raw files can be realized by wrapping a vendor-provided advanced programming interface (API) for the purpose of metadata analysis and visualization. Our novel package rawrr now provides complete, OS-independent access to all spectral data logged in Thermo Fisher Scientific raw files. In this technical note, we present implementation details and describe the main functionalities provided by the rawrr package. In addition, we report two use cases inspired by real-world research tasks that demonstrate the application of the package. The raw data used for demonstration purposes was deposited as MassIVE data set MSV000086542. Availability: https://github.com/fgcz/rawrr.
    Keywords:  R package; computational mass spectrometry; software
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00866
  29. Cell Death Dis. 2021 Mar 11. 12(3): 258
    Metabolomic analyses of COVID-19 patients unravel stage-dependent and prognostic biomarkers.
    François-Xavier Danlos, Claudia Grajeda-Iglesias, Sylvère Durand, Allan Sauvat, Mathilde Roumier, Delphine Cantin, Emeline Colomba, Julien Rohmer, Fanny Pommeret, Giulia Baciarello, Christophe Willekens, Marc Vasse, Frank Griscelli, Jean-Eudes Fahrner, Anne-Gaëlle Goubet, Agathe Dubuisson, Lisa Derosa, Nitharsshini Nirmalathasan, Delphine Bredel, Séverine Mouraud, Caroline Pradon, Annabelle Stoclin, Flore Rozenberg, Jérôme Duchemin, Georges Jourdi, Syrine Ellouze, Françoise Levavasseur, Laurence Albigès, Jean-Charles Soria, Fabrice Barlesi, Eric Solary, Fabrice André, Frédéric Pène, Félix Ackerman, Luc Mouthon, Laurence Zitvogel, Aurélien Marabelle, Jean-Marie Michot, Michaela Fontenay, Guido Kroemer.
      The circulating metabolome provides a snapshot of the physiological state of the organism responding to pathogenic challenges. Here we report alterations in the plasma metabolome reflecting the clinical presentation of COVID-19 patients with mild (ambulatory) diseases, moderate disease (radiologically confirmed pneumonitis, hospitalization and oxygen therapy), and critical disease (in intensive care). This analysis revealed major disease- and stage-associated shifts in the metabolome, meaning that at least 77 metabolites including amino acids, lipids, polyamines and sugars, as well as their derivatives, were altered in critical COVID-19 patient's plasma as compared to mild COVID-19 patients. Among a uniformly moderate cohort of patients who received tocilizumab, only 10 metabolites were different among individuals with a favorable evolution as compared to those who required transfer into the intensive care unit. The elevation of one single metabolite, anthranilic acid, had a poor prognostic value, correlating with the maintenance of high interleukin-10 and -18 levels. Given that products of the kynurenine pathway including anthranilic acid have immunosuppressive properties, we speculate on the therapeutic utility to inhibit the rate-limiting enzymes of this pathway including indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase.
    DOI:  https://doi.org/10.1038/s41419-021-03540-y
  30. J Proteome Res. 2021 Mar 08.
    Binary Classifier for Computing Posterior Error Probabilities in MetaMorpheus.
    Michael R Shortreed, Robert J Millikin, Lei Liu, Zach Rolfs, Rachel M Miller, Leah V Schaffer, Brian L Frey, Lloyd M Smith.
      MetaMorpheus is a free, open-source software program for the identification of peptides and proteoforms from data-dependent acquisition tandem MS experiments. There is inherent uncertainty in these assignments for several reasons, including the limited overlap between experimental and theoretical peaks, the m/z uncertainty, and noise peaks or peaks from coisolated peptides that produce false matches. False discovery rates provide only a set-wise approximation for incorrect spectrum matches. Here we implemented a binary decision tree calculation within MetaMorpheus to compute a posterior error probability, which provides a measure of uncertainty for each peptide-spectrum match. We demonstrate its utility for increasing identifications and resolving ambiguities in bottom-up, top-down, proteogenomic, and nonspecific digestion searches.
    Keywords:  DDA; MetaMorpheus; binary decision tree; bottom-up; open source; posterior error probability; proteogenomics; proteomics; search engine; top-down
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00838
  31. J Proteome Res. 2021 Mar 11.
    leapR: An R Package for Multiomic Pathway Analysis.
    Vincent Danna, Hugh Mitchell, Lindsey Anderson, Iobani Godinez, Sara J C Gosline, Justin Teeguarden, Jason E McDermott.
      A generalized goal of many high-throughput data studies is to identify functional mechanisms that underlie observed biological phenomena, whether they be disease outcomes or metabolic output. Increasingly, studies that rely on multiple sources of high-throughput data (genomic, transcriptomic, proteomic, metabolomic) are faced with a challenge of summarizing the data to generate testable hypotheses. However, this requires a time-consuming process to evaluate numerous statistical methods across numerous data sources. Here, we introduce the leapR package, a framework to rapidly assess biological pathway activity using diverse statistical tests and data sources, allowing facile integration of multisource data. The leapR package with a user manual and example workflow is available for download from GitHub (https://github.com/biodataganache/leapR).
    Keywords:  data integration; pathway analysis; phosphoproteomics; proteomics
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00963
  32. J Proteome Res. 2021 Mar 08.
    PHONEMeS: Efficient Modeling of Signaling Networks Derived from Large-Scale Mass Spectrometry Data.
    Enio Gjerga, Aurelien Dugourd, Luis Tobalina, Abel Sousa, Julio Saez-Rodriguez.
      Post-translational modifications of proteins play an important role in the regulation of cellular processes. The mass spectrometry analysis of proteome modifications offers huge potential for the study of how protein inhibitors affect the phosphosignaling mechanisms inside the cells. We have recently proposed PHONEMeS, a method that uses high-content shotgun phosphoproteomic data to build logical network models of signal perturbation flow. However, in its original implementation, PHONEMeS was computationally demanding and was only used to model signaling in a perturbation context. We have reformulated PHONEMeS as an Integer Linear Program (ILP) that is orders of magnitude more efficient than the original one. We have also expanded the scenarios that can be analyzed. PHONEMeS can model data upon perturbation on not only a known target but also deregulated pathways upstream and downstream of any set of deregulated kinases. Finally, PHONEMeS can now analyze data sets with multiple time points, which helps us to obtain better insight into the dynamics of the propagation of signals. We illustrate the value of the new approach on various data sets of medical relevance, where we shed light on signaling mechanisms and drug modes of action.
    Keywords:  cell signaling; integer linear programming; modelling; phosphoproteomics
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00958
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