bims-mascan 2020-03-08 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2020‒03‒08
twenty-two papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

Bacteria Boost Mammalian Host NAD Metabolism by Engaging the Deamidated Biosynthesis Pathway.
DJ-1 suppresses ferroptosis through preserving the activity of S-adenosyl homocysteine hydrolase.
Preparative supercritical fluid chromatography for lipid class fractionation-a novel strategy in high-resolution mass spectrometry based lipidomics.
Metabolic Plasticity of Tumor Cells: How They Do Adapt to Food Deprivation.
Strategies for drawing quantitative conclusions from non-targeted liquid chromatography high-resolution mass spectrometry analysis.
Metabolism in the tumor microenvironment: insights from single-cell analysis.
Metabolic Remodeling as a Way of Adapting to Tumor Microenvironment (TME), a Job of Several Holders.
Methionine Restriction and Cancer Biology.
Metabolic characterisation of THP-1 macrophage polarisation using LC-MS-based metabolite profiling.
Targeting metabolic activity in high-risk neuroblastoma through Monocarboxylate Transporter 1 (MCT1) inhibition.
Identification of the urine and serum metabolomics signature of gout.
Comprehensive Evaluation of a Quantitative Shotgun Lipidomics Platform for Mammalian Sample Analysis on a High-Resolution Mass Spectrometer.
Serum amino acid metabolic profiles of ankylosing spondylitis by targeted metabolomics analysis.
Metabolite Shifts Induced by Marathon Race Competition Differ between Athletes Based on Level of Fitness and Performance: A Substudy of the Enzy-MagIC Study.
The role of ubiquitination in tumorigenesis and targeted drug discovery.
Strategy for Global Profiling and Identification of 2- and 3-Hydroxy Fatty Acids in Plasma by UPLC-MS/MS.
Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy.
Appetite for Arginine: Metabolic Control of Macrophage Hunger.
Implementation of liquid chromatography-high resolution mass spectrometry methods for untargeted metabolomic analyses of biological samples: A tutorial.
Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress.
Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.
A high-throughput platform for detailed lipidomic analysis of a range of mouse and human tissues.

Cell Metab. 2020 Mar 03. pii: S1550-4131(20)30059-0. [Epub ahead of print]31(3): 564-579.e7

Bacteria Boost Mammalian Host NAD Metabolism by Engaging the Deamidated Biosynthesis Pathway.

Igor Shats, Jason G Williams, Juan Liu, Mikhail V Makarov, Xiaoyue Wu, Fred B Lih, Leesa J Deterding, Chaemin Lim, Xiaojiang Xu, Thomas A Randall, Ethan Lee, Wenling Li, Wei Fan, Jian-Liang Li, Marina Sokolsky, Alexander V Kabanov, Leping Li, Marie E Migaud, Jason W Locasale, Xiaoling Li.

  Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

Keywords:  NAMPT inhibitors; cancer cells; deamidated NAD synthesis; germ-free mice; host-microbe interaction; microbial nicotinamidase; mycoplasma; nicotinic acid; oral nicotinamide riboside supplementation

DOI:  https://doi.org/10.1016/j.cmet.2020.02.001
Nat Commun. 2020 Mar 06. 11(1): 1251

DJ-1 suppresses ferroptosis through preserving the activity of S-adenosyl homocysteine hydrolase.

Ji Cao, Xiaobing Chen, Li Jiang, Bin Lu, Meng Yuan, Difeng Zhu, Hong Zhu, Qiaojun He, Bo Yang, Meidan Ying.

Ferroptosis is a newly characterized form of regulated cell death mediated by iron-dependent accumulation of lipid reactive oxygen species and holds great potential for cancer therapy. However, the molecular mechanisms underlying ferroptosis remain largely elusive. In this study, we define an integrative role of DJ-1 in ferroptosis. Inhibition of DJ-1 potently enhances the sensitivity of tumor cells to ferroptosis inducers both in vitro and in vivo. Metabolic analysis and metabolite rescue assay reveal that DJ-1 depletion inhibits the transsulfuration pathway by disrupting the formation of the S-adenosyl homocysteine hydrolase tetramer and impairing its activity. Consequently, more ferroptosis is induced when homocysteine generation is decreased, which might be the only source of glutathione biosynthesis when cystine uptake is blocked. Thus, our findings show that DJ-1 determines the response of cancer cells to ferroptosis, and highlight a candidate therapeutic target to potentially improve the effect of ferroptosis-based antitumor therapy.

DOI: https://doi.org/10.1038/s41467-020-15109-y
Anal Bioanal Chem. 2020 Mar 04.

Preparative supercritical fluid chromatography for lipid class fractionation-a novel strategy in high-resolution mass spectrometry based lipidomics.

Harald Schoeny, Evelyn Rampler, Gerrit Hermann, Ulrike Grienke, Judith M Rollinger, Gunda Koellensperger.

  In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level. Graphical Abstract.

Keywords:  Human plasma; Lipid fractionation; Lipidomics; Pichia pastoris; Preparative supercritical fluid chromatography; SRM 1950

DOI:  https://doi.org/10.1007/s00216-020-02463-5
Adv Exp Med Biol. 2020 ;1219 109-123

Metabolic Plasticity of Tumor Cells: How They Do Adapt to Food Deprivation.

Céline A Schoonjans, Bernard Gallez.

  Dysregulated metabolism is a key hallmark of cancer cells and an enticing target for cancer treatment. Since the last 10 years, research on cancer metabolism has moved from pathway attention to network consideration. This metabolic complexity continuously adapt to new constraints in the tumor microenvironment. In this review, we will highlight striking changes in cancer cell metabolism compared to normal cells. Understanding this tumor metabolic plasticity suggests potential new targets and innovative combinatorial treatments for fighting cancer.

Keywords:  Acidosis; Amino acid; Bioenergetics; Combined therapies; Fatty acid; Glycolysis; Hypoxia; Metabolic reprogramming; Mitochondria

DOI:  https://doi.org/10.1007/978-3-030-34025-4_6
Anal Chem. 2020 Mar 05.

Strategies for drawing quantitative conclusions from non-targeted liquid chromatography high-resolution mass spectrometry analysis.

Anneli Kruve.

About ten years ago most of the liquid chromatography (LC) electrospray (ESI) mass spectrometry (MS) analysis of environmental, metabolomics, pharmaceutical samples were predominantly carried out as targeted analysis. Targeted analyses allow detection and quantification a few selected analytes with the aid of standard substances. Today, the centre of gravity is shifting towards non-targeted methods which utilize high-resolution mass spectrometry (HRMS). Furthermore, the targeted and non-targeted methods are merging into each other. LC/HRMS based non-targeted methods allow detecting compounds recovered from the sample preparation and ionizing in the electrospray ionization (ESI) source. New possibilities arising from applying the machine learning tools to LC/HRMS data have already transformed the process of identifying the compounds. The computer-aided identification process is not compatible with traditional calibration graph based quantification methods. The main obstacle arises from the fact that in ESI different compounds ionize to a very different extent. The differences up to 100 million times have been reported. This phenomenon results in a vastly different response of different compounds at the same concentration and complicates the quantification for compounds without standard substances. However, decision making is hindered without quantitative information. Therefore, the need to obtain quantitative information from the non-targeted analysis is triggering an emerging field of research. This review aims at giving an overview of different possibilities for quantitatively comparing the results obtained from LC/HRMS based non-targeted analysis. More specifically, quantification via structurally similar internal standards, different isotope labelling strategies, radiolabelling, and predicted ionization efficiencies are reviewed.

DOI: https://doi.org/10.1021/acs.analchem.9b03481
Oncoimmunology. 2020 ;9(1): 1726556

Metabolism in the tumor microenvironment: insights from single-cell analysis.

Zhengtao Xiao, Jason W Locasale, Ziwei Dai.

  The metabolism of both cancer and immune cells in the tumor microenvironment (TME) is poorly understood since most studies have focused on analysis in bulk samples and ex vivo cell culture models. Our recent analyses of single-cell RNA sequencing data suggest that the metabolic features of single cells within TME differ greatly from those of the bulk measurements. Here, we discuss some key findings about metabolism in cancer and immune cells and discuss possible relevance to immunotherapy.

Keywords:  Cancer metabolism; immunometabolism; single-cell RNA sequencing; tumor microenvironment

DOI:  https://doi.org/10.1080/2162402X.2020.1726556
Adv Exp Med Biol. 2020 ;1219 1-34

Metabolic Remodeling as a Way of Adapting to Tumor Microenvironment (TME), a Job of Several Holders.

Jacinta Serpa.

  The microenvironment depends and generates dependence on all the cells and structures that share the same niche, the biotope. The contemporaneous view of the tumor microenvironment (TME) agrees with this idea. The cells that make up the tumor, whether malignant or not, behave similarly to classes of elements within a living community. These elements inhabit, modify and benefit from all the facilities the microenvironment has to offer and that will contribute to the survival and growth of the tumor and the progression of the disease.The metabolic adaptation to microenvironment is a crucial process conducting to an established tumor able to grow locally, invade and metastasized. The metastatic cancer cells are reasonable more plastic than non-metastatic cancer cells, because the previous ones must survive in the microenvironment where the primary tumor develops and in addition, they must prosper in the microenvironment in the metastasized organ.The metabolic remodeling requires not only the adjustment of metabolic pathways per se but also the readjustment of signaling pathways that will receive and obey to the extracellular instructions, commanding the metabolic adaptation. Many diverse players are pivotal in cancer metabolic fitness from the initial signaling stimuli, going through the activation or repression of genes, until the phenotype display. The new phenotype will permit the import and consumption of organic compounds, useful for energy and biomass production, and the export of metabolic products that are useless or must be secreted for a further recycling or controlled uptake. In the metabolic network, three subsets of players are pivotal: (1) the organic compounds; (2) the transmembrane transporters, and (3) the enzymes.This chapter will present the "Pharaonic" intent of diagraming the interplay between these three elements in an attempt of simplifying and, at the same time, of showing the complex sight of cancer metabolism, addressing the orchestrating role of microenvironment and highlighting the influence of non-cancerous cells.

Keywords:  Cancer cell metabolism; Fatty acids synthesis; Glutaminolysis; Glycolysis; Metabolic network; Metabolic remodeling; One-carbon metabolism; Pentose phosphate pathway (PPP); Transsulfuration pathway (TSSP); Tumor microenvironment (TME); β-oxidation

DOI:  https://doi.org/10.1007/978-3-030-34025-4_1
Nutrients. 2020 Mar 03. pii: E684. [Epub ahead of print]12(3):

Methionine Restriction and Cancer Biology.

Desiree Wanders, Katherine Hobson, Xiangming Ji.

  The essential amino acid, methionine, is important for cancer cell growth and metabolism. A growing body of evidence indicates that methionine restriction inhibits cancer cell growth and may enhance the efficacy of chemotherapeutic agents. This review summarizes the efficacy and mechanism of action of methionine restriction on hallmarks of cancer in vitro and in vivo. The review highlights the role of glutathione formation, polyamine synthesis, and methyl group donation as mediators of the effects of methionine restriction on cancer biology. The translational potential of the use of methionine restriction as a personalized nutritional approach for the treatment of patients with cancer is also discussed.

Keywords:  amino acids; cancer; glutathione; methionine; methylation

DOI:  https://doi.org/10.3390/nu12030684
Metabolomics. 2020 Feb 29. 16(3): 33

Metabolic characterisation of THP-1 macrophage polarisation using LC-MS-based metabolite profiling.

Alaa Abuawad, Chidimma Mbadugha, Amir M Ghaemmaghami, Dong-Hyun Kim.

  INTRODUCTION: Macrophages constitute a heterogeneous population of functionally distinct cells involved in several physiological and pathological processes. They display remarkable plasticity by changing their phenotype and function in response to environmental cues representing a spectrum of different functional phenotypes. The so-called M1 and M2 macrophages are often considered as representative of pro- and anti-inflammatory ends of such spectrum. Metabolomics approach is a powerful tool providing important chemical information about the cellular phenotype of living systems, and the changes in their metabolic pathways in response to various perturbations.OBJECTIVES: This study aimed to characterise M1 and M2 phenotypes in THP-1 macrophages in order to identify characteristic metabolites of each polarisation state.
METHODS: Herein, untargeted liquid chromatography (LC)-mass spectrometry (MS)-based metabolite profiling was applied to characterise the metabolic profile of M1-like and M2-like THP-1 macrophages.
RESULTS: The results showed that M1 and M2 macrophages have distinct metabolic profiles. Sphingolipid and pyrimidine metabolism was significantly changed in M1 macrophages whereas arginine, proline, alanine, aspartate and glutamate metabolism was significantly altered in M2 macrophages.
CONCLUSION: This study represents successful application of LC-MS metabolomics approach to characterise M1 and M2 macrophages providing functional readouts that show unique metabolic signature for each phenotype. These data could contribute to a better understanding of M1 and M2 functional properties and could pave the way for developing new therapeutics targeting different immune diseases.

Keywords:  LC–MS; Macrophage polarisation; Metabolic pathway analysis; Metabolite profiling; THP-1 cell

DOI:  https://doi.org/10.1007/s11306-020-01656-4
Oncogene. 2020 Mar 02.

Targeting metabolic activity in high-risk neuroblastoma through Monocarboxylate Transporter 1 (MCT1) inhibition.

Aaminah Khan, Emanuele Valli, Hayley Lam, David A Scott, Jayne Murray, Kimberley M Hanssen, Georgina Eden, Laura D Gamble, Rupinder Pandher, Claudia L Flemming, Sophie Allan, Andrei L Osterman, Michelle Haber, Murray D Norris, Jamie I Fletcher, Denise M T Yu.

Amplification of the MYCN oncogene occurs in ~25% of primary neuroblastomas and is the single most powerful biological marker of poor prognosis in this disease. MYCN transcriptionally regulates a range of biological processes important for cancer, including cell metabolism. The MYCN-regulated metabolic gene SLC16A1, encoding the lactate transporter monocarboxylate transporter 1 (MCT1), is a potential therapeutic target. Treatment of neuroblastoma cells with the MCT1 inhibitor SR13800 increased intracellular lactate levels, disrupted the nicotinamide adenine dinucleotide (NADH/NAD+) ratio, and decreased intracellular glutathione levels. Metabolite tracing with 13C-glucose and 13C-glutamine following MCT1 inhibitor treatment revealed increased quantities of tricarboxylic acid (TCA) cycle intermediates and increased oxygen consumption rate. MCT1 inhibition was highly synergistic with vincristine and LDHA inhibition under cell culture conditions, but this combination was ineffective against neuroblastoma xenografts. Posttreatment xenograft tumors had increased synthesis of the MCT1 homolog MCT4/SLC16A, a known resistance factor to MCT1 inhibition. We found that MCT4 was negatively regulated by MYCN in luciferase reporter assays and its synthesis in neuroblastoma cells was increased under hypoxic conditions and following hypoxia-inducible factor (HIF1) induction, suggesting that MCT4 may contribute to resistance to MCT1 inhibitor treatment in hypoxic neuroblastoma tumors. Co-treatment of neuroblastoma cells with inhibitors of MCT1 and LDHA, the enzyme responsible for lactate production, resulted in a large increase in intracellular pyruvate and was highly synergistic in decreasing neuroblastoma cell viability. These results highlight the potential of targeting MCT1 in neuroblastoma in conjunction with strategies that involve disruption of pyruvate homeostasis and indicate possible resistance mechanisms.

DOI: https://doi.org/10.1038/s41388-020-1235-2
Rheumatology (Oxford). 2020 Mar 05. pii: keaa018. [Epub ahead of print]

Identification of the urine and serum metabolomics signature of gout.

Yefei Huang, Min Xiao, Jiayong Ou, Qing Lv, Qiujing Wei, Zena Chen, Jialing Wu, Liudan Tu, Yutong Jiang, Xi Zhang, Jun Qi, Minli Qiu, Shuangyan Cao, Jieruo Gu.

  OBJECTIVE: Gout is the most common inflammatory arthritis and the worldwide incidence is increasing. By revealing the metabolic alterations in serum and urine of gout patients, the first aim of our study was to discover novel molecular biomarkers allowing for early diagnosis. We also aimed to investigate the underlying pathogenic pathways.METHODS: Serum and urine samples from gout patients (n = 30) and age-matched healthy controls (n = 30) were analysed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to screen the differential metabolites and construct a diagnostic model. Next, the model was verified and optimized in the second validation cohort (n = 100). The pathways were illustrated to understand the underlying pathogenesis of gout.
RESULTS: In general, serum metabolomics demonstrated a clearer distinction than urine metabolomics. In the discovery cohort, 40 differential serum metabolites were identified that could distinguish gout patients from healthy controls. Among them, eight serum metabolites were verified in the validation cohort. Through regression analysis, the final model consisted of three serum metabolites-pyroglutamic acid, 2-methylbutyryl carnitine and Phe-Phe-that presented optimal diagnostic power. The three proposed metabolites produced an area under the curve of 0.956 (95% CI 0.911, 1.000). Additionally, the proposed metabolic pathways were primarily involved in purine metabolism, branched-chain amino acids (BCAAs) metabolism, the tricarboxylic acid cycle, synthesis and degradation of ketone bodies, bile secretion and arachidonic acid metabolism.
CONCLUSION: The metabolomics signatures could serve as an efficient tool for early diagnosis and provide novel insights into the pathogenesis of gout.

Keywords:  biomarkers; branched-chain amino acids; gout; metabolomics; uric acid

DOI:  https://doi.org/10.1093/rheumatology/keaa018
J Am Soc Mass Spectrom. 2020 Mar 04.

Comprehensive Evaluation of a Quantitative Shotgun Lipidomics Platform for Mammalian Sample Analysis on a High-Resolution Mass Spectrometer.

Inger Ødum Nielsen, André Vidas Olsen, Jano Dicroce-Giacobini, Elena Papaleo, Klaus Kaae Andersen, Marja Jäättelä, Kenji Maeda, Mesut Bilgin.

  Shotgun lipidomics is a powerful tool that enables simultaneous and fast quantification of diverse lipid classes through mass spectrometry based analyses of directly infused crude lipid extracts. We present here a shotgun lipidomics platform established to quantify 38 lipid classes belonging to four lipid categories present in mammalian samples and show the fine-tuning and comprehensive evaluation of its experimental parameters and performance. We first determined for all the targeted lipid classes the collision energy levels optimal for the recording of their lipid class- and species-specific fragment ions and fine-tuned the energy levels applied in the platform. We then performed a series of titrations to define the boundaries of linear signal response for the targeted lipid classes, and demonstrated that the dynamic quantification range spanned more than 3 orders of magnitude and reached sub picomole levels for 35 lipid classes. The platform identified 273, 261, and 287 lipid species in brain, plasma, and cultured fibroblast samples, respectively, at the respective optimal working sample amounts. The platform properly quantified the majority of these identified lipid species, while lipid species measured to be below the limit of quantification were efficiently removed from the data sets by the use of statistical analyses of data reproducibility or a cutoff threshold. Finally, we demonstrated that a series of parameters of cell culture conditions influence lipidomics outcomes, including confluency, medium supplements, and use of transfection reagents. The present study provides a guideline for setting up and using a simple and efficient platform for quantitatively exploring the mammalian lipidome.

Keywords:  evaluation; high-resolution mass spectrometry; mammalian lipidome; shotgun lipidomics

DOI:  https://doi.org/10.1021/jasms.9b00136
Clin Rheumatol. 2020 Mar 04.

Serum amino acid metabolic profiles of ankylosing spondylitis by targeted metabolomics analysis.

Yong Zhou, Xi Zhang, Rui Chen, Su Han, Yishu Liu, Xuefeng Liu, Ming Gao, Chundong Yang, Daifeng Lu, Beibei Sun, Hao Chen.

  BACKGROUND: Ankylosing spondylitis (AS) is a common chronic inflammatory arthritis, causing lasting back pain with progressive loss of spinal mobility. However, the exact pathogenesis of AS remains unclear. We aim to use the metabolomics analysis to characterize the metabolic profile of AS, in order to better understand the pathogenesis of AS and monitor disease activity and progression.METHODS: The ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQ-MS) was used for investigating the serum amino acid metabolomic profiling of 30 AS patients, in comparison with 32 rheumatoid arthritis (RA) patients and 30 healthy controls, combined with multivariate statistical analysis. Metabolite association analysis with disease activity was performed using generalized linear regression. The metabolic pathway analysis for the important metabolites was performed using MetPA and the metabolic network was constructed.
RESULTS: A total of 29 amino acids and biogenic amines were detected in all participants by UPLC-TQ-MS. It showed significant amino acid differences between the AS/RA patients and control subjects. Additionally, 4-hydroxy-L-proline, alanine, γ-aminobutyric acid, glutamine, and taurine were identified as candidate markers shared by AS/RA groups. Specifically, lysine, proline, serine, and alanine were found correlated with disease activity of AS. Furthermore, the most significant metabolic pathway identified were alanine, aspartate, and glutamate metabolism, arginine and proline metabolism, aminoacyl tRNA biosynthesis and glycine, serine, and threonine metabolism.
CONCLUSIONS: These preliminary results demonstrate that UPLC-TQ-MS analysis method is a powerful tool to identify metabolite profiles of AS. Research in identified disease activity-associated metabolites and biological pathways may provide assistance for clinical diagnosis and pathological mechanism of AS.Key Points• There are perturbations of serum amino acid metabolism in AS, compared with RA and healthy controls, determined by UPLC-TQ-MS.• Metabolomics pathway is used to analysis for the differential metabolites of AS.• The altered serum amino acid could monitor disease activity of AS.

Keywords:  Amino acids; Ankylosing spondylitis; Biomarker; UPLC-TQ-MS

DOI:  https://doi.org/10.1007/s10067-020-04974-z
Metabolites. 2020 Mar 01. pii: E87. [Epub ahead of print]10(3):

Metabolite Shifts Induced by Marathon Race Competition Differ between Athletes Based on Level of Fitness and Performance: A Substudy of the Enzy-MagIC Study.

Jana F Schader, Mark Haid, Alexander Cecil, Julia Schoenfeld, Martin Halle, Arne Pfeufer, Cornelia Prehn, Jerzy Adamski, David C Nieman, Johannes Scherr.

  This study compared metabolite shifts induced by training for, participation in, and recovery from a marathon race competition among athletes divided into three groups based on fitness (relative maximum oxygen uptake (VO2max)) and performance levels (net running time). Plasma samples from 76 male runners participating in the Munich Marathon were analyzed for metabolite shifts using a targeted metabolomics panel. For the entire cohort of runners, pronounced increases were measured immediately after the race for plasma concentrations of acylcarnitines (AC), the ratio (palmitoylcarnitine + stearoylcarnitine)/free carnitine that is used as a proxy for the activity of the mitochondrial enzyme carnitine palmitoyltransferase, and arginine-related metabolites, with decreases in most amino acids (AA) and phospholipids. Plasma levels of AA and phospholipids were strongly increased 24 and 72 h post-race. Post-race plasma concentrations of AC and arginine-related metabolites were higher in the low compared to top performers, indicating an accumulation of fatty acids and a reliance on protein catabolism to provide energy after the marathon event. This study showed that marathon race competition is associated with an extensive and prolonged perturbation in plasma metabolite concentrations with a strong AC signature that is greater in the slower, less aerobically fit runners. Furthermore, changes in the arginine-related metabolites were observed.

Keywords:  amino acids; biomarker; exercise; fatty acids; metabolism; urea cycle

DOI:  https://doi.org/10.3390/metabo10030087
Signal Transduct Target Ther. 2020 ;5 11

The role of ubiquitination in tumorigenesis and targeted drug discovery.

Lu Deng, Tong Meng, Lei Chen, Wenyi Wei, Ping Wang.

  Ubiquitination, an important type of protein posttranslational modification (PTM), plays a crucial role in controlling substrate degradation and subsequently mediates the "quantity" and "quality" of various proteins, serving to ensure cell homeostasis and guarantee life activities. The regulation of ubiquitination is multifaceted and works not only at the transcriptional and posttranslational levels (phosphorylation, acetylation, methylation, etc.) but also at the protein level (activators or repressors). When regulatory mechanisms are aberrant, the altered biological processes may subsequently induce serious human diseases, especially various types of cancer. In tumorigenesis, the altered biological processes involve tumor metabolism, the immunological tumor microenvironment (TME), cancer stem cell (CSC) stemness and so on. With regard to tumor metabolism, the ubiquitination of some key proteins such as RagA, mTOR, PTEN, AKT, c-Myc and P53 significantly regulates the activity of the mTORC1, AMPK and PTEN-AKT signaling pathways. In addition, ubiquitination in the TLR, RLR and STING-dependent signaling pathways also modulates the TME. Moreover, the ubiquitination of core stem cell regulator triplets (Nanog, Oct4 and Sox2) and members of the Wnt and Hippo-YAP signaling pathways participates in the maintenance of CSC stemness. Based on the altered components, including the proteasome, E3 ligases, E1, E2 and deubiquitinases (DUBs), many molecular targeted drugs have been developed to combat cancer. Among them, small molecule inhibitors targeting the proteasome, such as bortezomib, carfilzomib, oprozomib and ixazomib, have achieved tangible success. In addition, MLN7243 and MLN4924 (targeting the E1 enzyme), Leucettamol A and CC0651 (targeting the E2 enzyme), nutlin and MI-219 (targeting the E3 enzyme), and compounds G5 and F6 (targeting DUB activity) have also shown potential in preclinical cancer treatment. In this review, we summarize the latest progress in understanding the substrates for ubiquitination and their special functions in tumor metabolism regulation, TME modulation and CSC stemness maintenance. Moreover, potential therapeutic targets for cancer are reviewed, as are the therapeutic effects of targeted drugs.

Keywords:  Cancer metabolism; Cancer microenvironment; Cancer stem cells; Cancer therapy

DOI:  https://doi.org/10.1038/s41392-020-0107-0
Anal Chem. 2020 Mar 05.

Strategy for Global Profiling and Identification of 2- and 3-Hydroxy Fatty Acids in Plasma by UPLC-MS/MS.

Jiangshuo Li, Jing Xu, Ruiping Zhang, Yanzeng Hao, Jiuming He, Yanhua Chen, Guanggen Jiao, Zeper Abliz.

2-Hydroxy fatty acids (2-OHFAs) and 3-hydroxy fatty acids (3-OHFAs) with the same carbon backbone are isomers, both of which are closely related to diseases involving fatty acid oxidation disorder. However, the comprehensive profiling of 2- and 3-OHFAs remains an ongoing challenge due to their high structure similarity, few structure-informative fragment ions and limited availability of standards. Here, we developed a new strategy to profile and identify 2- and 3-OHFAs according to structure-dependent retention time prediction models using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Both accurate MS and MS/MS spectra were collected for peak annotation by comparison with an in-house database of theoretically possible 2- and 3-OHFAs. The structures were further confirmed by the validated structure-dependent retention time prediction models, taking advantage of the correlation between the retention time, carbon chain length and number of double bonds, as well as the hydroxyl position-induced isomeric retention time shift rule. Using this strategy, eighteen 2-OHFAs and thirty-two 3-OHFAs were identified in the pooled plasma, of which seven 2-OHFAs and twenty 3-OHFAs are identified for the first time in this work, furthering our understanding of OHFA metabolism. Subsequent quantitation method was developed by scheduled multiple reaction monitoring (MRM), and then applied to investigate the alteration of 2- and 3-OHFAs in esophageal squamous cell carcinoma (ESCC) patients. Finally, a potential biomarker panel consisted of 6 OHFAs with good diagnostic performance was achieved. Our study provides a new strategy for isomer identification and analysis, showing a great potential for targeted metabolomics in clinical biomarker discovery.

DOI: https://doi.org/10.1021/acs.analchem.9b05627
J Biol Chem. 2020 Mar 05. pii: jbc.RA119.012376. [Epub ahead of print]

Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy.

Renee C Geck, Jackson R Foley, Tracy Murray Stewart, John M Asara, Robert A Casero, Alex Toker.

  Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was due to a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator, antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced, preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Keywords:  DNA damage; amino acid; antizyme; breast cancer; difluoromethylornithine; metabolomics; ornithine decarboxylase (ODC); polyamine; triple-negative breast cancer (TNBC)

DOI:  https://doi.org/10.1074/jbc.RA119.012376
Cell Metab. 2020 Mar 03. pii: S1550-4131(20)30064-4. [Epub ahead of print]31(3): 441-442

Appetite for Arginine: Metabolic Control of Macrophage Hunger.

Daniel J Puleston, Erika L Pearce.

Engulfment of dying cells by phagocytes is essential to maintain tissue function and promote injury resolution and repair. This process, termed efferocytosis, requires persistent corpse engulfment and remains a poorly understood mechanism. Here, we preview findings from Yurdagul et al. (2020) that detail how continual efferocytosis is supported by metabolites derived from the dying cell itself.

DOI: https://doi.org/10.1016/j.cmet.2020.02.005
Anal Chim Acta. 2020 Apr 08. pii: S0003-2670(19)31532-6. [Epub ahead of print]1105 28-44

Implementation of liquid chromatography-high resolution mass spectrometry methods for untargeted metabolomic analyses of biological samples: A tutorial.

Julian Pezzatti, Julien Boccard, Santiago Codesido, Yoric Gagnebin, Abhinav Joshi, Didier Picard, Víctor González-Ruiz, Serge Rudaz.

  Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. By its nature, this is a highly interdisciplinary field of research, and mastering all of the steps comprised in the pipeline can be a challenging task, especially for those researchers new to the topic. In this tutorial, we aim to provide an overview of the most widely adopted methods of performing LC-HRMS-based untargeted metabolomics of biological samples. A detailed protocol is provided in the Supplementary Information for rapidly implementing a basic screening workflow in a laboratory setting. This tutorial covers experimental design, sample preparation and analysis, signal processing and data treatment, and, finally, data analysis and its biological interpretation. Each section is accompanied by up-to-date literature to guide readers through the preparation and optimization of such a workflow, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls.

Keywords:  Biological samples; High-resolution mass spectrometry; Liquid chromatography; Metabolomics; Methodology; Untargeted

DOI:  https://doi.org/10.1016/j.aca.2019.12.062
EMBO J. 2020 Mar 05. e103334

Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress.

Simon Schwörer, Mirela Berisa, Sara Violante, Weige Qin, Jiajun Zhu, Ronald C Hendrickson, Justin R Cross, Craig B Thompson.

  The production and secretion of matrix proteins upon stimulation of fibroblasts by transforming growth factor-beta (TGFβ) play a critical role in wound healing. How TGFβ supports the bioenergetic cost of matrix protein synthesis is not fully understood. Here, we show that TGFβ promotes protein translation at least in part by increasing the mitochondrial oxidation of glucose and glutamine carbons to support the bioenergetic demand of translation. Surprisingly, we found that in addition to stimulating the entry of glucose and glutamine carbon into the TCA cycle, TGFβ induced the biosynthesis of proline from glutamine in a Smad4-dependent fashion. Metabolic manipulations that increased mitochondrial redox generation promoted proline biosynthesis, while reducing mitochondrial redox potential and/or ATP synthesis impaired proline biosynthesis. Thus, proline biosynthesis acts as a redox vent, preventing the TGFβ-induced increase in mitochondrial glucose and glutamine catabolism from generating damaging reactive oxygen species (ROS) when TCA cycle activity exceeds the ability of oxidative phosphorylation to convert mitochondrial redox potential into ATP. In turn, the enhanced synthesis of proline supports TGFβ-induced production of matrix proteins.

Keywords:  TGFβ; collagen; fibrosis; metabolism; proline

DOI:  https://doi.org/10.15252/embj.2019103334
Nat Methods. 2020 Mar 02.

Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.

Jared O Kafader, Rafael D Melani, Kenneth R Durbin, Bon Ikwuagwu, Bryan P Early, Ryan T Fellers, Steven C Beu, Vlad Zabrouskov, Alexander A Makarov, Joshua T Maze, Deven L Shinholt, Ping F Yip, Danielle Tullman-Ercek, Michael W Senko, Philip D Compton, Neil L Kelleher.

An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.

DOI: https://doi.org/10.1038/s41592-020-0764-5
Anal Bioanal Chem. 2020 Mar 07.

A high-throughput platform for detailed lipidomic analysis of a range of mouse and human tissues.

Samuel Furse, Denise S Fernandez-Twinn, Benjamin Jenkins, Claire L Meek, Huw E L Williams, Gordon C S Smith, D Stephen Charnock-Jones, Susan E Ozanne, Albert Koulman.

  Lipidomics is of increasing interest in studies of biological systems. However, high-throughput data collection and processing remains non-trivial, making assessment of phenotypes difficult. We describe a platform for surveying the lipid fraction for a range of tissues. These techniques are demonstrated on a set of seven different tissues (serum, brain, heart, kidney, adipose, liver, and vastus lateralis muscle) from post-weaning mouse dams that were either obese (> 12 g fat mass) or lean (<5 g fat mass). This showed that the lipid metabolism in some tissues is affected more by obesity than others. Analysis of human serum (healthy non-pregnant women and pregnant women at 28 weeks' gestation) showed that the abundance of several phospholipids differed between groups. Human placenta from mothers with high and low BMI showed that lean placentae contain less polyunsaturated lipid. This platform offers a way to map lipid metabolism with immediate application in metabolic research and elsewhere. Graphical abstract.

Keywords:  31P NMR; Human development; Lipid profiling; Lipidomics; Mass spectrometry; Metabolic disease; Mouse model

DOI:  https://doi.org/10.1007/s00216-020-02511-0