bims-mascan 2021-01-31 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2021–01–31
24 papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh

Lack of Electron Acceptors Contributes to Redox Stress and Growth Arrest in Asparagine-Starved Sarcoma Cells.
Spatial differentiation of metabolism in prostate cancer tissue by MALDI-TOF MSI.
Metabolism of Amino Acids in Cancer.
Shared reference materials harmonize lipidomics across MS-based detection platforms and laboratories.
Ferroptosis: mechanisms, biology and role in disease.
Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry.
Separation and identification of permethylated glycan isomers by reversed phase nanoLC-NSI-MSn.
G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.
TNF-α induces acyl-CoA synthetase 3 to promote lipid droplet formation in human endothelial cells.
Metabolic Regulation of Ferroptosis in Cancer.
Beyond glucose: alternative sources of energy in glioblastoma.
The potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models.
Lipidome-wide 13C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures.
A Validated Method for Quantification of Fatty Acids Incorporated in Human Plasma Phospholipids by Gas Chromatography-Triple Quadrupole Mass Spectrometry.
Upregulated ethanolamine phospholipid synthesis by selenoprotein I is required for effective metabolic reprogramming during T cell activation.
Targeted Metabolomics Identifies Plasma Biomarkers in Mice with Metabolically Heterogeneous Melanoma Xenografts.
Different ion mobility-mass spectrometry coupling techniques to promote metabolomics.
Pathway Tools Visualization of Organism-Scale Metabolic Networks.
The ins and outs of serine and glycine metabolism in cancer.
Reviewing the metabolome coverage provided by LC-MS: Focus on sample preparation and chromatography-A tutorial.
Isolation and characterization of extracellular vesicle subpopulations from tissues.
Targeted eicosanoids profiling reveals a prostaglandin reprogramming in breast Cancer by microRNA-155.
Statistical Approach to Protein Quantification.
Serine, glycine and one‑carbon metabolism in cancer (Review).

Cancers (Basel). 2021 Jan 22. pii: 412. [Epub ahead of print]13(3):

Lack of Electron Acceptors Contributes to Redox Stress and Growth Arrest in Asparagine-Starved Sarcoma Cells.

Christoph Bauer, Meret Quante, Willemijn B Breunis, Carla Regina, Michaela Schneider, Geoffroy Andrieux, Oliver Gorka, Olaf Groß, Melanie Boerries, Bernd Kammerer, Simone Hettmer.

  Amino acids are integral components of cancer metabolism. The non-essential amino acid asparagine supports the growth and survival of various cancer cell types. Here, different mass spectrometry approaches were employed to identify lower aspartate levels, higher aspartate/glutamine ratios and lower tricarboxylic acid (TCA) cycle metabolite levels in asparagine-deprived sarcoma cells. Reduced nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide hydride (NADH) ratios were consistent with redirection of TCA cycle flux and relative electron acceptor deficiency. Elevated lactate/pyruvate ratios may be due to compensatory NAD+ regeneration through increased pyruvate to lactate conversion by lactate dehydrogenase. Supplementation with exogenous pyruvate, which serves as an electron acceptor, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Chemicals disrupting NAD+ regeneration in the electron transport chain further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. We speculate that reductive stress may be a major contributor to the growth arrest observed in asparagine-starved cells.

Keywords:  asparagine starvation; metabolomics; reductive stress; sarcoma

DOI:  https://doi.org/10.3390/cancers13030412
Cancer Metab. 2021 Jan 29. 9(1): 9

Spatial differentiation of metabolism in prostate cancer tissue by MALDI-TOF MSI.

Maria K Andersen, Therese S Høiem, Britt S R Claes, Benjamin Balluff, Marta Martin-Lorenzo, Elin Richardsen, Sebastian Krossa, Helena Bertilsson, Ron M A Heeren, Morten B Rye, Guro F Giskeødegård, Tone F Bathen, May-Britt Tessem.

   BACKGROUND: Prostate cancer tissues are inherently heterogeneous, which presents a challenge for metabolic profiling using traditional bulk analysis methods that produce an averaged profile. The aim of this study was therefore to spatially detect metabolites and lipids on prostate tissue sections by using mass spectrometry imaging (MSI), a method that facilitates molecular imaging of heterogeneous tissue sections, which can subsequently be related to the histology of the same section.
METHODS: Here, we simultaneously obtained metabolic and lipidomic profiles in different prostate tissue types using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI. Both positive and negative ion mode were applied to analyze consecutive sections from 45 fresh-frozen human prostate tissue samples (N = 15 patients). Mass identification was performed with tandem MS.
RESULTS: Pairwise comparisons of cancer, non-cancer epithelium, and stroma revealed several metabolic differences between the tissue types. We detected increased levels of metabolites crucial for lipid metabolism in cancer, including metabolites involved in the carnitine shuttle, which facilitates fatty acid oxidation, and building blocks needed for lipid synthesis. Metabolites associated with healthy prostate functions, including citrate, aspartate, zinc, and spermine had lower levels in cancer compared to non-cancer epithelium. Profiling of stroma revealed higher levels of important energy metabolites, such as ADP, ATP, and glucose, and higher levels of the antioxidant taurine compared to cancer and non-cancer epithelium.
CONCLUSIONS: This study shows that specific tissue compartments within prostate cancer samples have distinct metabolic profiles and pinpoint the advantage of methodology providing spatial information compared to bulk analysis. We identified several differential metabolites and lipids that have potential to be developed further as diagnostic and prognostic biomarkers for prostate cancer. Spatial and rapid detection of cancer-related analytes showcases MALDI-TOF MSI as a promising and innovative diagnostic tool for the clinic.

Keywords:  Mass spectrometry imaging; Metabolism; Prostate cancer; Tumor heterogeneity

DOI:  https://doi.org/10.1186/s40170-021-00242-z
Front Cell Dev Biol. 2020 ;8 603837

Metabolism of Amino Acids in Cancer.

Zhen Wei, Xiaoyi Liu, Chunming Cheng, Wei Yu, Ping Yi.

  Metabolic reprogramming has been widely recognized as a hallmark of malignancy. The uptake and metabolism of amino acids are aberrantly upregulated in many cancers that display addiction to particular amino acids. Amino acids facilitate the survival and proliferation of cancer cells under genotoxic, oxidative, and nutritional stress. Thus, targeting amino acid metabolism is becoming a potential therapeutic strategy for cancer patients. In this review, we will systematically summarize the recent progress of amino acid metabolism in malignancy and discuss their interconnection with mammalian target of rapamycin complex 1 (mTORC1) signaling, epigenetic modification, tumor growth and immunity, and ferroptosis. Finally, we will highlight the potential therapeutic applications.

Keywords:  amino acids (AAs); cancer; epigenetic; ferroptosis; mTORC (mammalian target of rapamycin kinase complex); metabolism; tumor growth; tumor immunity

DOI:  https://doi.org/10.3389/fcell.2020.603837
J Lipid Res. 2020 Jan;pii: S0022-2275(20)30020-1. [Epub ahead of print]61(1): 105-115

Shared reference materials harmonize lipidomics across MS-based detection platforms and laboratories.

Alexander Triebl, Bo Burla, Jayashree Selvalatchmanan, Jeongah Oh, Sock Hwee Tan, Mark Y Chan, Natalie A Mellet, Peter J Meikle, Federico Torta, Markus R Wenk.

  Quantitative MS of human plasma lipids is a promising technology for translation into clinical applications. Current MS-based lipidomic methods rely on either direct infusion (DI) or chromatographic lipid separation methods (including reversed phase and hydrophilic interaction LC). However, the use of lipid markers in laboratory medicine is limited by the lack of reference values, largely because of considerable differences in the concentrations measured by different laboratories worldwide. These inconsistencies can be explained by the use of different sample preparation protocols, method-specific calibration procedures, and other experimental and data-reporting parameters, even when using identical starting materials. Here, we systematically investigated the roles of some of these variables in multiple approaches to lipid analysis of plasma samples from healthy adults by considering: 1) different sample introduction methods (separation vs. DI methods); 2) different MS instruments; and 3) between-laboratory differences in comparable analytical platforms. Each of these experimental variables resulted in different quantitative results, even with the inclusion of isotope-labeled internal standards for individual lipid classes. We demonstrated that appropriate normalization to commonly available reference samples (i.e., "shared references") can largely correct for these systematic method-specific quantitative biases. Thus, to harmonize data in the field of lipidomics, in-house long-term references should be complemented by a commonly available shared reference sample, such as NIST SRM 1950, in the case of human plasma.

Keywords:  National Institute of Standards and Technology standard reference material 1950; harmonization; lipids; liquid chromatography; mass spectrometry; plasma; quantitation

DOI:  https://doi.org/10.1194/jlr.D119000393
Nat Rev Mol Cell Biol. 2021 Jan 25.

Ferroptosis: mechanisms, biology and role in disease.

Xuejun Jiang, Brent R Stockwell, Marcus Conrad.

The research field of ferroptosis has seen exponential growth over the past few years, since the term was coined in 2012. This unique modality of cell death, driven by iron-dependent phospholipid peroxidation, is regulated by multiple cellular metabolic pathways, including redox homeostasis, iron handling, mitochondrial activity and metabolism of amino acids, lipids and sugars, in addition to various signalling pathways relevant to disease. Numerous organ injuries and degenerative pathologies are driven by ferroptosis. Intriguingly, therapy-resistant cancer cells, particularly those in the mesenchymal state and prone to metastasis, are exquisitely vulnerable to ferroptosis. As such, pharmacological modulation of ferroptosis, via both its induction and its inhibition, holds great potential for the treatment of drug-resistant cancers, ischaemic organ injuries and other degenerative diseases linked to extensive lipid peroxidation. In this Review, we provide a critical analysis of the current molecular mechanisms and regulatory networks of ferroptosis, the potential physiological functions of ferroptosis in tumour suppression and immune surveillance, and its pathological roles, together with a potential for therapeutic targeting. Importantly, as in all rapidly evolving research areas, challenges exist due to misconceptions and inappropriate experimental methods. This Review also aims to address these issues and to provide practical guidelines for enhancing reproducibility and reliability in studies of ferroptosis. Finally, we discuss important concepts and pressing questions that should be the focus of future ferroptosis research.

DOI: https://doi.org/10.1038/s41580-020-00324-8
J Vis Exp. 2021 Jan 05.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Karamat Mohammad, Heng Jiang, Vladimir I Titorenko.

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/µL. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

DOI: https://doi.org/10.3791/62061
Mol Cell Proteomics. 2021 Jan 25. pii: S1535-9476(20)35174-4. [Epub ahead of print] 100045

Separation and identification of permethylated glycan isomers by reversed phase nanoLC-NSI-MSn.

Simone Kurz, M Osman Sheikh, Shan Lu, Lance Wells, Michael Tiemeyer.

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

DOI: https://doi.org/10.1074/mcp.RA120.002266
Cell. 2021 Jan 18. pii: S0092-8674(20)31694-9. [Epub ahead of print]

G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling.

Genomics England Research Consortium

  Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Keywords:  G3BP1; G3BP2; TSC complex; cancer; lysosome; mTORC1; metabolism; neuronal function; stress granule

DOI:  https://doi.org/10.1016/j.cell.2020.12.024
J Lipid Res. 2020 Jan;pii: S0022-2275(20)30014-6. [Epub ahead of print]61(1): 33-44

TNF-α induces acyl-CoA synthetase 3 to promote lipid droplet formation in human endothelial cells.

Hye Seung Jung, Masami Shimizu-Albergine, Xia Shen, Farah Kramer, Dan Shao, Anuradha Vivekanandan-Giri, Subramaniam Pennathur, Rong Tian, Jenny E Kanter, Karin E Bornfeldt.

  Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.

Keywords:  cytokines; fatty acid/metabolism; fatty acid/oxidation

DOI:  https://doi.org/10.1194/jlr.RA119000256
Biology (Basel). 2021 Jan 22. pii: 83. [Epub ahead of print]10(2):

Metabolic Regulation of Ferroptosis in Cancer.

Min Ji Kim, Greg Jiho Yun, Sung Eun Kim.

  Ferroptosis is a unique cell death mechanism that is executed by the excessive accumulation of lipid peroxidation in cells. The relevance of ferroptosis in multiple human diseases such as neurodegeneration, organ damage, and cancer is becoming increasingly evident. As ferroptosis is deeply intertwined with metabolic pathways such as iron, cyst(e)ine, glutathione, and lipid metabolism, a better understanding of how ferroptosis is regulated by these pathways will enable the precise utilization or prevention of ferroptosis for therapeutic uses. In this review, we present an update of the mechanisms underlying diverse metabolic pathways that can regulate ferroptosis in cancer.

Keywords:  GPX4; SLC7A11; cyst(e)ine metabolism; ferroptosis; glutathione metabolism; iron metabolism; lipid peroxidation; reactive oxygen species

DOI:  https://doi.org/10.3390/biology10020083
Theranostics. 2021 ;11(5): 2048-2057

Beyond glucose: alternative sources of energy in glioblastoma.

John L Caniglia, Anvesh Jalasutram, Swapna Asuthkar, Joseph Sahagun, Simon Park, Aditya Ravindra, Andrew J Tsung, Maheedhara R Guda, Kiran K Velpula.

  Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. With a designation of WHO Grade IV, it is also the most lethal primary brain tumor with a median survival of just 15 months. This is often despite aggressive treatment that includes surgical resection, radiation therapy, and chemotherapy. Based on the poor outcomes and prevalence of the tumor, the demand for innovative therapies continues to represent a pressing issue for clinicians and researchers. In terms of therapies targeting metabolism, the prevalence of the Warburg effect has led to a focus on targeting glucose metabolism to halt tumor progression. While glucose is the dominant source of growth substrate in GBM, a number of unique metabolic pathways are exploited in GBM to meet the increased demand for replication and progression. In this review we aim to explore how metabolites from fatty acid oxidation, the urea cycle, the glutamate-glutamine cycle, and one-carbon metabolism are shunted toward energy producing pathways to meet the high energy demand in GBM. We will also explore how the process of autophagy provides a reservoir of nutrients to support viable tumor cells. By so doing, we aim to establish a foundation of implicated metabolic mechanisms supporting growth and tumorigenesis of GBM within the literature. With the sparse number of therapeutic interventions specifically targeting metabolic pathways in GBM, we hope that this review expands further insight into the development of novel treatment modalities.

Keywords:  arginine; autophagy; fatty acids; glioblastoma; glutamine; metabolism

DOI:  https://doi.org/10.7150/thno.53506
Cell Oncol (Dordr). 2021 Jan 25.

The potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models.

Clara Lemos, Volker K Schulze, Simon J Baumgart, Ekaterina Nevedomskaya, Tobias Heinrich, Julien Lefranc, Benjamin Bader, Clara D Christ, Hans Briem, Lara P Kuhnke, Simon J Holton, Ulf Bömer, Philip Lienau, Franz von Nussbaum, Carl F Nising, Marcus Bauser, Andrea Hägebarth, Dominik Mumberg, Bernard Haendler.

   PURPOSE: 5' adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor.
METHODS: High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation.
RESULTS: We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux.
CONCLUSIONS: The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.

Keywords:  AMPK; Androgen signaling; Lipid metabolism; Prostate cancer

DOI:  https://doi.org/10.1007/s13402-020-00584-8
J Lipid Res. 2020 Jan;pii: S0022-2275(20)30019-5. [Epub ahead of print]61(1): 95-104

Lipidome-wide 13C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures.

Michael Schlame, Yang Xu, Hediye Erdjument-Bromage, Thomas A Neubert, Mindong Ren.

  Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, because it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are often used as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of 13C6-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with 13C6-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homolog of the calcium-independent phospholipase A2β, on lipid metabolism. Although 13C6-glucose gave rise to a complex pattern of 13C incorporation, we were able to identify discrete isotopomers in which 13C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.

Keywords:  genes in lipid dysfunction; lipid metabolism; mass spectrometry; phospholipids/metabolism; stable isotope tracers

DOI:  https://doi.org/10.1194/jlr.D119000318
ACS Omega. 2021 Jan 19. 6(2): 1129-1137

A Validated Method for Quantification of Fatty Acids Incorporated in Human Plasma Phospholipids by Gas Chromatography-Triple Quadrupole Mass Spectrometry.

Hans-F Schött, Maurice C J M Konings, Vera B Schrauwen-Hinderling, Ronald P Mensink, Jogchum Plat.

Fatty acids (FA) are important mediators of health maintenance and disease risk. Optimal quantification assays of FA in high and low abundance as well the identification of 13C-labeled tracers to monitor FA metabolism are of major interest. The article on hand reports about the development and validation of a gas chromatography (GC)-triple quadrupole mass selective detection (GC-TQMS) method for absolute quantification of FA in human plasma phospholipids (hpPL). The quantification of the calibration solution by GC-flame ionization detection (GC-FID), with the introduction of a correction factor, allows the direct comparison of individual FA concentrations in hpPL by GC-TQMS. Specificity, sensitivity, and reproducibility are achieved by optimized chromatographic separation and employment of GC-TQMS. The inter-method comparison between GC-FID and GC-TQMS concentrations revealed good comparability for 27 FA. A full validation has been performed with linearity over 4 magnitudes, a limit of detection of 0.18-38.3 fmol on column, a recovery of 83.6-109.6%, and intraday and interday precision data meeting the criteria of EMA and FDA guidelines. The method includes the absolute quantification of 58 positional and geometrical (cis/trans) isomeric FA in hpPL in the concentration range of 1-3000 nmol/mL, covering also low abundant positional cis/trans isomers. Results obtained from both methods are highly comparable, and selectivity and sensitivity are improved by using GC-TQMS. Additionally, we show here that calculation of 13C-labeled C16:0 tracer/tracee ratios in hpPL in human isotope enrichment studies is possible.

DOI: https://doi.org/10.1021/acsomega.0c03874
Mol Metab. 2021 Jan 20. pii: S2212-8778(21)00010-7. [Epub ahead of print] 101170

Upregulated ethanolamine phospholipid synthesis by selenoprotein I is required for effective metabolic reprogramming during T cell activation.

Chi Ma, FuKun W Hoffmann, Michael P Marciel, Kathleen E Page, Melodie A Williams-Aduja, Ellis N L Akana, Greg S Gojanovich, Mariana Gerschenson, Johann Urschitz, Stefan Moisyadi, Vedbar S Khadka, Sharon Rozovsky, Youping Deng, F David Horgen, Peter R Hoffmann.

   OBJECTIVE: T cell activation triggers metabolic reprogramming to meet increased demand for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cells and cancer cells, which led us to investigate a potential role during T cell activation.
METHODS: Since selenoprotein I (SELENOI) is an enzyme participating in two metabolic pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation.
RESULTS: SELENOI knockout (KO) in mouse T cells led to reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor, AMPK. AMPK is inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment.
CONCLUSIONS: T cell activation upregulates SELENOI-dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.

Keywords:  AMPK; ethanolamine phosphotransferase; glycolysis; metabolic sensing; phosphatidylethanolamine; selenium; selenoprotein

DOI:  https://doi.org/10.1016/j.molmet.2021.101170
Cancers (Basel). 2021 Jan 23. pii: 434. [Epub ahead of print]13(3):

Targeted Metabolomics Identifies Plasma Biomarkers in Mice with Metabolically Heterogeneous Melanoma Xenografts.

Daniela D Weber, Maheshwor Thapa, Sepideh Aminzadeh-Gohari, Anna-Sophia Redtenbacher, Luca Catalano, René G Feichtinger, Peter Koelblinger, Guido Dallmann, Michael Emberger, Barbara Kofler, Roland Lang.

  Melanomas are genetically and metabolically heterogeneous, which influences therapeutic efficacy and contributes to the development of treatment resistance in patients with metastatic disease. Metabolite phenotyping helps to better understand complex metabolic diseases, such as melanoma, and facilitates the development of novel therapies. Our aim was to characterize the tumor and plasma metabolomes of mice bearing genetically different melanoma xenografts. We engrafted the human melanoma cell lines A375 (BRAF mutant), WM47 (BRAF mutant), WM3000 (NRAS mutant), and WM3311 (BRAF, NRAS, NF1 triple-wildtype) and performed a broad-spectrum targeted metabolomics analysis of tumor and plasma samples obtained from melanoma-bearing mice as well as plasma samples from healthy control mice. Differences in ceramide and phosphatidylcholine species were observed between melanoma subtypes irrespective of the genetic driver mutation. Furthermore, beta-alanine metabolism differed between melanoma subtypes and was significantly enriched in plasma from melanoma-bearing mice compared to healthy mice. Moreover, we identified beta-alanine, p-cresol sulfate, sarcosine, tiglylcarnitine, two dihexosylceramides, and one phosphatidylcholine as potential melanoma biomarkers in plasma. The present data reflect the metabolic heterogeneity of melanomas but also suggest a diagnostic biomarker signature for melanoma screening.

Keywords:  Warburg effect; beta-alanine metabolism; cancer metabolism; lipid metabolism; melanoma; metabolic biomarker; targeted metabolomics

DOI:  https://doi.org/10.3390/cancers13030434
Mass Spectrom Rev. 2021 Jan 25.

Different ion mobility-mass spectrometry coupling techniques to promote metabolomics.

Aurélie Delvaux, Estelle Rathahao-Paris, Sandra Alves.

  Metabolomics has become increasingly popular in recent years for many applications ranging from clinical diagnosis, human health to biotechnological questioning. Despite technological advances, metabolomic studies are still currently limited by the difficulty of identifying all metabolites, a class of compounds with great chemical diversity. Although lengthy chromatographic analyses are often used to obtain comprehensive data, many isobar and isomer metabolites still remain unresolved, which is a critical point for the compound identification. Currently, ion mobility spectrometry is being explored in metabolomics as a way to improve metabolome coverage, analysis throughput and isomer separation. In this review, all the steps of a typical workflow for untargeted metabolomics are discussed considering the use of an ion mobility instrument. An overview of metabolomics is first presented followed by a brief description of ion mobility instrumentation. The ion mobility potential for complex mixture analysis is discussed regarding its coupling with a mass spectrometer alone, providing gas-phase separation before mass analysis as well as its combination with different separation platforms (conventional hyphenation but also multidimensional ion mobility couplings), offering multidimensional separation. Various instrumental and analytical conditions for improving the ion mobility separation are also described. Finally, data mining, including software packages and visualization approaches, as well as the construction of ion mobility databases for the metabolite identification are examined.

Keywords:  Ion mobility-mass spectrometry; hyphenated method; metabolomics; multidimensional data

DOI:  https://doi.org/10.1002/mas.21685
Metabolites. 2021 Jan 22. pii: 64. [Epub ahead of print]11(2):

Pathway Tools Visualization of Organism-Scale Metabolic Networks.

Suzanne Paley, Richard Billington, James Herson, Markus Krummenacker, Peter D Karp.

  Metabolomics, synthetic biology, and microbiome research demand information about organism-scale metabolic networks. The convergence of genome sequencing and computational inference of metabolic networks has enabled great progress toward satisfying that demand by generating metabolic reconstructions from the genomes of thousands of sequenced organisms. Visualization of whole metabolic networks is critical for aiding researchers in understanding, analyzing, and exploiting those reconstructions. We have developed bioinformatics software tools that automatically generate a full metabolic-network diagram for an organism, and that enable searching and analyses of the network. The software generates metabolic-network diagrams for unicellular organisms, for multi-cellular organisms, and for pan-genomes and organism communities. Search tools enable users to find genes, metabolites, enzymes, reactions, and pathways within a diagram. The diagrams are zoomable to enable researchers to study local neighborhoods in detail and to see the big picture. The diagrams also serve as tools for comparison of metabolic networks and for interpreting high-throughput datasets, including transcriptomics, metabolomics, and reaction fluxes computed by metabolic models. These data can be overlaid on the metabolic charts to produce animated zoomable displays of metabolic flux and metabolite abundance. The BioCyc.org website contains whole-network diagrams for more than 18,000 sequenced organisms. The ready availability of organism-specific metabolic network diagrams and associated tools for almost any sequenced organism are useful for researchers working to better understand the metabolism of their organism and to interpret high-throughput datasets in a metabolic context.

Keywords:  biochemical pathways charts; metabolic charts; metabolic diagrams; metabolic maps; metabolic network diagrams; metabolomics; transcriptomics

DOI:  https://doi.org/10.3390/metabo11020064
Nat Metab. 2021 Jan 28.

The ins and outs of serine and glycine metabolism in cancer.

Shauni L Geeraerts, Elien Heylen, Kim De Keersmaecker, Kim R Kampen.

Cancer cells reprogramme their metabolism to support unrestrained proliferation and survival in nutrient-poor conditions. Whereas non-transformed cells often have lower demands for serine and glycine, several cancer subtypes hyperactivate intracellular serine and glycine synthesis and become addicted to de novo production. Copy-number amplifications of serine- and glycine-synthesis genes and genetic alterations in common oncogenes and tumour-suppressor genes enhance serine and glycine synthesis, resulting in high production and secretion of these oncogenesis-supportive metabolites. In this Review, we discuss the contribution of serine and glycine synthesis to cancer progression. By relying on de novo synthesis pathways, cancer cells are able to enhance macromolecule synthesis, neutralize high levels of oxidative stress and regulate methylation and tRNA formylation. Furthermore, we discuss the immunosuppressive potential of serine and glycine, and the essentiality of both amino acids to promoting survival of non-transformed neighbouring cells. Finally, we point to the emerging data proposing moonlighting functions of serine- and glycine-synthesis enzymes and examine promising small molecules targeting serine and glycine synthesis.

DOI: https://doi.org/10.1038/s42255-020-00329-9
Anal Chim Acta. 2021 Feb 22. pii: S0003-2670(20)31218-6. [Epub ahead of print]1147 38-55

Reviewing the metabolome coverage provided by LC-MS: Focus on sample preparation and chromatography-A tutorial.

Marta Roca, Maria Isabel Alcoriza, Juan Carlos Garcia-Cañaveras, Agustín Lahoz.

  Metabolomics has become an invaluable tool for both studying metabolism and biomarker discovery. The great technical advances in analytical chemistry and bioinformatics have considerably increased the number of measurable metabolites, yet an important part of the human metabolome remains uncovered. Among the various MS hyphenated techniques available, LC-MS stands out as the most used. Here, we aimed to show the capabilities of LC-MS to uncover part of the metabolome and how to best proceed with sample preparation and LC to maximise metabolite detection. The analyses of various open metabolite databases served us to estimate the size of the already detected human metabolome, the expected metabolite composition of most used human biospecimens and which part of the metabolome can be detected when LC-MS is used. Based on an extensive review and on our experience, we have outlined standard procedures for LC-MS analysis of urine, cells, serum/plasma, tissues and faeces, to guide in the selection of the sample preparation method that best matches with one or more LC techniques in order to get the widest metabolome coverage. These standard procedures may be a useful tool to explore, at a glance, the wide spectrum of possibilities available, which can be a good starting point for most of the LC-MS metabolomic studies.

Keywords:  Biospecimens; Human metabolome; LC-MS; Metabolome coverage; Metabolomics

DOI:  https://doi.org/10.1016/j.aca.2020.12.025
Nat Protoc. 2021 Jan 25.

Isolation and characterization of extracellular vesicle subpopulations from tissues.

Rossella Crescitelli, Cecilia Lässer, Jan Lötvall.

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

DOI: https://doi.org/10.1038/s41596-020-00466-1
J Exp Clin Cancer Res. 2021 Jan 25. 40(1): 43

Targeted eicosanoids profiling reveals a prostaglandin reprogramming in breast Cancer by microRNA-155.

Sinae Kim, Eun Sung Lee, Eun Ji Lee, Jae Yun Jung, Sae Byul Lee, Hee Jin Lee, Jisun Kim, Hee Jeong Kim, Jong Won Lee, Byung Ho Son, Gyungyub Gong, Sei-Hyun Ahn, Suhwan Chang.

   BACKGROUND: Prostaglandin is one of the key metabolites for inflammation-related carcinogenesis. Despite the microRNA-155 is implicated in various types of cancers, it's function in prostaglandin metabolism is largely unknown.
METHODS: A targeted profiling of eicosanoids including prostaglandin, leukotriene and thromboxanes was performed in miR-155 deficient breast tumors and cancer cells. The molecular mechanism of miR-155-mediated prostaglandin reprogramming was investigated in primary and cancer cell lines, by analyzing key enzymes responsible for the prostaglandin production.
RESULTS: We found miR-155-deficient breast tumors, plasma of tumor-bearing mouse and cancer cells show altered prostaglandin level, especially for the prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2). Subsequent analysis in primary cancer cells, 20 triple-negative breast cancer (TNBC) specimens and breast cancer cell lines with miR-155 knockdown consistently showed a positive correlation between miR-155 level and PGE2/PGD2 ratio. Mechanistically, we reveal the miR-155 reprograms the prostaglandin metabolism by up-regulating PGE2-producing enzymes PTGES/PTGES2 while down-regulating PGD2-producing enzyme PTGDS. Further, we show the up-regulation of PTGES2 is driven by miR-155-cMYC axis, whereas PTGES is transactivated by miR-155-KLF4. Thus, miR-155 hires dual-regulatory mode for the metabolic enzyme expression to reprogram the PGE2/PGD2 balance. Lastly, we show the miR-155-driven cellular proliferation is restored by the siRNA of PTGES1/2, of which expression also significantly correlates with breast cancer patients' survival.
CONCLUSIONS: Considering clinical trials targeting PGE2 production largely have focused on the inhibition of Cox1 or Cox2 that showed cardiac toxicity, our data suggest an alternative way for suppressing PGE2 production via the inhibition of miR-155. As the antagomiR of miR-155 (MRG-106) underwent a phase-1 clinical trial, its effect should be considered and analyzed in prostaglandin metabolism in tumor.

Keywords:  KLF4; Microrna-155; Myc; PTGES1; PTGES2; Prostaglandin D2; Prostaglandin E2; TNBC

DOI:  https://doi.org/10.1186/s13046-021-01839-4
Mol Cell Proteomics. 2014 Feb;pii: S1535-9476(20)34652-1. [Epub ahead of print]13(2): 666-677

Statistical Approach to Protein Quantification.

Sarah Gerster, Taejoon Kwon, Christina Ludwig, Mariette Matondo, Christine Vogel, Edward M Marcotte, Ruedi Aebersold, Peter Bühlmann.

A major goal in proteomics is the comprehensive and accurate description of a proteome. This task includes not only the identification of proteins in a sample, but also the accurate quantification of their abundance. Although mass spectrometry typically provides information on peptide identity and abundance in a sample, it does not directly measure the concentration of the corresponding proteins. Specifically, most mass-spectrometry-based approaches (e.g. shotgun proteomics or selected reaction monitoring) allow one to quantify peptides using chromatographic peak intensities or spectral counting information. Ultimately, based on these measurements, one wants to infer the concentrations of the corresponding proteins. Inferring properties of the proteins based on experimental peptide evidence is often a complex problem because of the ambiguity of peptide assignments and different chemical properties of the peptides that affect the observed concentrations. We present SCAMPI, a novel generic and statistically sound framework for computing protein abundance scores based on quantified peptides. In contrast to most previous approaches, our model explicitly includes information from shared peptides to improve protein quantitation, especially in eukaryotes with many homologous sequences. The model accounts for uncertainty in the input data, leading to statistical prediction intervals for the protein scores. Furthermore, peptides with extreme abundances can be reassessed and classified as either regular data points or actual outliers. We used the proposed model with several datasets and compared its performance to that of other, previously used approaches for protein quantification in bottom-up mass spectrometry.

DOI: https://doi.org/10.1074/mcp.M112.025445
Int J Oncol. 2020 Dec 11.

Serine, glycine and one‑carbon metabolism in cancer (Review).

Sijing Pan, Ming Fan, Zhangnan Liu, Xia Li, Huijuan Wang.

Serine/glycine biosynthesis and one‑carbon metabolism are crucial in sustaining cancer cell survival and rapid proliferation, and of high clinical relevance. Excessive activation of serine/glycine biosynthesis drives tumorigenesis and provides a single carbon unit for one‑carbon metabolism. One‑carbon metabolism, which is a complex cyclic metabolic network based on the chemical reaction of folate compounds, provides the necessary proteins, nucleic acids, lipids and other biological macromolecules to support tumor growth. Moreover, one‑carbon metabolism also maintains the redox homeostasis of the tumor microenvironment and provides substrates for the methylation reaction. The present study reviews the role of key enzymes with tumor‑promoting functions and important intermediates that are physiologically relevant to tumorigenesis in serine/glycine/one‑carbon metabolism pathways. The related regulatory mechanisms of action of the key enzymes and important intermediates in tumors are also discussed. It is hoped that investigations into these pathways will provide new translational opportunities for human cancer drug development, dietary interventions, and biomarker identification.

DOI: https://doi.org/10.3892/ijo.2020.5158