bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2022‒11‒20
34 papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. J Am Soc Mass Spectrom. 2022 Nov 15.
      Lipidomics has developed rapidly over the past decade. Nontargeted lipidomics from biological samples remains a challenge due to the high structural diversity, the concentration range of lipids, and the complexity of biological samples. We introduce here the use of differential Kendrick's plots as a rapid visualization tool for a qualitative nontargeted analysis of lipids categories and classes from data generated by either liquid chromatography-mass spectrometry (LC-MS) or direct infusion (nESI-MS). Each lipid class is easily identified by comparison with the theoretical Kendrick plot pattern constructed from exact mass measurements and by using MSKendrickFilter, an in-house Python software. The lipids are identified with the LIPID MAPS database. In addition, in LC-MS, the software based on the Kendrick plots returns the retention time from all the lipids belonging to the same series. Lipid extracts from a yeast (Saccharomyces cerevisiae) are used as a model. An on/off case comparing Kendrick plots from two cell lines (prostate cancer cell lines treated or not with a DGAT2 inhibition) clearly shows the effect of the inhibition. Our study demonstrates the good performance of direct infusion as a fast qualitative screening method as well as for the analysis of chromatograms. A fast screening semiquantitative approach is also possible, while the targeted mode remains the golden standard for precise quantitative analysis.
  2. Proteomics. 2022 Nov 18. e2200039
      Human plasma is a rich source of biomedical information and biomarkers. However, the enormous dynamic range of plasma proteins limits its accessibility to mass spectrometric (MS) analysis. Here, we show that enrichment of extracellular vesicles (EV) by ultracentrifugation increases plasma proteome depth by an order of magnitude. With this approach, more than two thousand proteins are routinely and reproducibly quantified by label-free quantification and data independent acquisition (DIA) in single-shot liquid chromatography tandem mass spectrometry runs of less than one hour. We present an optimized plasma proteomics workflow that enables high-throughput with very short chromatographic gradients analyzing hundred samples per day with deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples. Finally, we test the workflow on clinical biobank samples from malignant melanoma patients in immunotherapy to demonstrate the improved proteome coverage supporting the potential for future biomarker discovery. This article is protected by copyright. All rights reserved.
    Keywords:  Biomarkers; Cancer Immune Therapy; Extracellular vesicles; Plasma proteomics
  3. Apoptosis. 2022 Nov 18.
      It has been 10 years since the concept of ferroptosis was put forward and research focusing on ferroptosis has been increasing continuously. Ferroptosis is driven by iron-dependent lipid peroxidation, which can be antagonized by glutathione peroxidase 4 (GPX4), ferroptosis inhibitory protein 1 (FSP1), dihydroorotate dehydrogenase (DHODH) and Fas-associated factor 1 (FAF1). Various cellular metabolic events, including lipid metabolism, can modulate ferroptosis sensitivity. It is worth noting that the reprogramming of lipid metabolism in cancer cells can promote the occurrence and development of tumors. The metabolic flexibility of cancer cells opens the possibility for the coordinated targeting of multiple lipid metabolic pathways to trigger cancer cells ferroptosis. In addition, cancer cells must obtain immortality, escape from programmed cell death including ferroptosis, to promote cancer progression, which provides new perspectives for improving cancer therapy. Targeting the vulnerability of ferroptosis has received attention as one of the significant possible strategies to treat cancer given its role in regulating tumor cell survival. We review the impact of iron and lipid metabolism on ferroptosis and the potential role of the crosstalk of lipid metabolism reprogramming and ferroptosis in antitumor immunity and sum up agents targeting lipid metabolism and ferroptosis for cancer therapy.
    Keywords:  Anti-tumor immunity; Ferroptosis; Ferroptotic cancer therapy; Lipid metabolism
  4. Proteomics. 2022 Nov 18. e2100308
      Membrane lipids play important roles in the regulation of cell fate, including the execution of ferroptosis. Ferroptosis is a non-apoptotic cell death mechanism defined by iron-dependent membrane lipid peroxidation. Phospholipids containing polyunsaturated fatty acids (PUFAs) are highly vulnerable to peroxidation and are essential for ferroptosis execution. By contrast, the incorporation of less oxidizable monounsaturated fatty acids (MUFAs) in membrane phospholipids protects cells from ferroptosis. The enzymes and pathways that govern PUFA and MUFA metabolism therefore play a critical role in determining cellular sensitivity to ferroptosis. Here, we review three lipid metabolic processes fatty acid biosynthesis, ether lipid biosynthesis, and phospholipid remodeling-that govern ferroptosis sensitivity by regulating the balance of PUFAs and MUFAs in membrane phospholipids. This article is protected by copyright. All rights reserved.
    Keywords:  PUFA; ether lipid; ferroptosis; iron; membrane; necrosis
  5. Cell Rep. 2022 Nov 15. pii: S2211-1247(22)01510-8. [Epub ahead of print]41(7): 111639
      T cells dynamically rewire their metabolism during an immune response. We applied single-cell RNA sequencing to CD8+ T cells activated and differentiated in vitro in physiological medium to resolve these metabolic dynamics. We identify a differential time-dependent reliance of activating T cells on the synthesis versus uptake of various non-essential amino acids, which we corroborate with functional assays. We also identify metabolic genes that potentially dictate the outcome of T cell differentiation, by ranking them based on their expression dynamics. Among them, we find asparagine synthetase (Asns), whose expression peaks for effector T cells and decays toward memory formation. Disrupting these expression dynamics by ASNS overexpression promotes an effector phenotype, enhancing the anti-tumor response of adoptively transferred CD8+ T cells in a mouse melanoma model. We thus provide a resource of dynamic expression changes during CD8+ T cell activation and differentiation, and identify ASNS expression dynamics as a modulator of CD8+ T cell differentiation.
    Keywords:  ASNS; CD8(+) T cells; CP: Immunology; T-cell activation; T-cell differentiation; asparagine; dynamics; immunology; metabolism; physiological media; scRNA-seq
  6. Analyst. 2022 Nov 16.
      Proteins are the key biological actors within cells, driving many biological processes integral to both healthy and diseased states. Understanding the depth of complexity represented within the proteome is crucial to our scientific understanding of cellular biology and to provide disease specific insights for clinical applications. Mass spectrometry-based proteomics is the premier method for proteome analysis, with the ability to both identify and quantify proteins. Although proteomics continues to grow as a robust field of bioanalytical chemistry, advances are still necessary to enable a more comprehensive view of the proteome. In this review, we provide a broad overview of mass spectrometry-based proteomics in general, and highlight four developing areas of bottom-up proteomics: (1) protein inference, (2) alternative proteases, (3) sample-specific databases and (4) post-translational modification discovery.
  7. Cancer Res Commun. 2022 Jul;2(7): 694-705
      Glutamine is the most abundant non-essential amino acid in blood stream; yet it's concentration in tumor interstitium is markedly lower than that in the serum, reflecting the huge demand of various cell types in tumor microenvironment for glutamine. While many studies have investigated glutamine metabolism in tumor epithelium and infiltrating immune cells, the role of glutamine metabolism in tumor blood vessels remains unknown. Here, we report that inducible genetic deletion of glutaminase (GLS) specifically in host endothelium, GLSECKO, impairs tumor growth and metastatic dissemination in vivo. Loss of GLS decreased tumor microvascular density, increased perivascular support cell coverage, improved perfusion, and reduced hypoxia in mammary tumors. Importantly, chemotherapeutic drug delivery and therapeutic efficacy were improved in tumor-bearing GLSECKO hosts or in combination with GLS inhibitor, CB839. Mechanistically, loss of GLS in tumor endothelium resulted in decreased leptin levels, and exogenous recombinant leptin rescued tumor growth defects in GLSECKO mice. Together, these data demonstrate that inhibition of endothelial glutamine metabolism normalizes tumor vessels, reducing tumor growth and metastatic spread, improving perfusion, and reducing hypoxia, and enhancing chemotherapeutic delivery. Thus, targeting glutamine metabolism in host vasculature may improve clinical outcome in patients with solid tumors.
    Keywords:  glutaminase; glutamine; host-tumor interactions; leptin; vessel normalization
  8. Clin Epigenetics. 2022 Nov 12. 14(1): 145
      Epigenetics includes a complex set of processes that alter gene activity without modifying the DNA sequence, which ultimately determines how the genetic information common to all the cells of an organism is used to generate different cell types. Dysregulation in the deposition and maintenance of epigenetic features, which include histone posttranslational modifications (PTMs) and histone variants, can result in the inappropriate expression or silencing of genes, often leading to diseased states, including cancer. The investigation of histone PTMs and variants in the context of clinical samples has highlighted their importance as biomarkers for patient stratification and as key players in aberrant epigenetic mechanisms potentially targetable for therapy. Mass spectrometry (MS) has emerged as the most powerful and versatile tool for the comprehensive, unbiased and quantitative analysis of histone proteoforms. In recent years, these approaches-which we refer to as "epi-proteomics"-have demonstrated their usefulness for the investigation of epigenetic mechanisms in pathological conditions, offering a number of advantages compared with the antibody-based methods traditionally used to profile clinical samples. In this review article, we will provide a critical overview of the MS-based approaches that can be employed to study histone PTMs and variants in clinical samples, with a strong focus on the latest advances in this area, such as the analysis of uncommon modifications and the integration of epi-proteomics data into multi-OMICs approaches, as well as the challenges to be addressed to fully exploit the potential of this novel field of research.
    Keywords:  Cancer; Epigenetics; Histone posttranslational modification; Histone variant; Histone-modifying enzyme; Mass spectrometry; Proteomics
  9. Anal Chem. 2022 Nov 17.
      Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107─108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.
  10. J Mass Spectrom Adv Clin Lab. 2022 Nov;26 28-33
      Background: Despite its clear advantages over immunoassay-based testing, the measurement of serum thyroglobulin by mass spectrometry remains limited to a handful of institutions. Slow adoption by clinical laboratories could reflect limited accessibility to existing methods that have sensitivity comparable to modern immunoassays, as well as a lack of tools for calibration and assay harmonization.Methods: We developed and validated a liquid chromatography-tandem mass spectrometry-based assay for the quantification of serum thyroglobulin. The protocol combined peptide immunoaffinity purification using a commercially available, well-characterized monoclonal antibody and mobile phase modification with dimethylsulfoxide (DMSO) for enhanced sensitivity. To facilitate harmonization with other laboratories, we developed a novel, serum-based 5-point distributable reference material (Husky Ref).
    Results: The assay demonstrated a lower limit of quantification of 0.15 ng/mL (<20 %CV). Mobile phase DMSO increased signal intensity of the target peptide at least 3-fold, improving quantification at low concentrations. Calibration traceable to Husky Ref enabled harmonization between laboratories in an interlaboratory study.
    Conclusions: Sensitive mass spectrometry-based thyroglobulin measurement can be achieved using a monoclonal antibody during peptide immunoaffinity purification and the addition of mobile phase DMSO. Laboratories interested in deploying this assay can utilize the provided standard operating procedure and freely-available Husky Ref reference material.
    Keywords:  Autoantibody; CPTAC, Clinical Proteomic Tumor Analysis Consortium; DMSO, dimethylsulfoxide; DTC, differentiated thyroid cancer; Differentiated thyroid cancer; IAE, immunoaffinity enrichment; LC-MS/MS, liquid chromatography-tandem mass spectrometry; Mass spectrometry; TLCK, tosyllysine chloromethyl ketone hydrochloride; Tg, thyroglobulin; Thyroglobulin
  11. Adv Sci (Weinh). 2022 Nov 16. e2202642
      Lacking a clear understanding of the molecular mechanism determining cancer cell sensitivity to oxidative phosphorylation (OXPHOS) inhibition limits the development of OXPHOS-targeting cancer treatment. Here, cancer cell lines sensitive or resistant to OXPHOS inhibition are identified by screening. OXPHOS inhibition-sensitive cancer cells possess increased OXPHOS activity and silenced nicotinamide N-methyltransferase (NNMT) expression. NNMT expression negatively correlates with OXPHOS inhibition sensitivity and functionally downregulates the intracellular levels of S-adenosyl methionine (SAM). Expression of DNA methyltransferase 1 (DNMT1), a SAM consumer, positively correlates with OXPHOS inhibition sensitivity. NNMT overexpression and DNMT1 inhibition render OXPHOS inhibition-sensitive cancer cells resistant. Importantly, treatments of OXPHOS inhibitors (Gboxin and Berberine) hamper the growth of mouse tumor xenografts by OXPHOS inhibition sensitive but not resistant cancer cells. What's more, the retrospective study of 62 tumor samples from a clinical trial demonstrates that administration of Berberine reduces the tumor recurrence rate of NNMTlow /DNMT1high but not NNMThigh /DNMT1low colorectal adenomas (CRAs). These results thus reveal a critical role of the NNMT-DNMT1 axis in determining cancer cell reliance on mitochondrial OXPHOS and suggest that NNMT and DNMT1 are faithful biomarkers for OXPHOS-targeting cancer therapies.
    Keywords:  DNA methylation; N-methyltransferase (NNMT); S-adenosyl methionine (SAM); biomarker; cancer; oxidative phosphorylation (OXPHOS)
  12. Methods Mol Biol. 2023 ;2596 399-419
      Assays for measuring enzyme activity can be useful tools for proteomics applications. Enzyme testing can be performed to validate an experimental system prior to a difference gel electrophoresis (DIGE) proteomic experiment and can also be utilized as an integral part of multifaceted experiment in conjunction with DIGE. Data from enzyme tests can be used to corroborate results of DIGE proteomic experiments where an enzyme or enzymes are demonstrated by DIGE to be differentially expressed. Enzyme testing can also be utilized to support data from DIGE experiments that demonstrate metabolic changes in a biological system. The different types of enzyme assays that can be performed in conjunction with DIGE experiments are reviewed alongside a discussion of experimental approaches for designing enzyme assays.
    Keywords:  Difference gel electrophoresis (DIGE); Enzyme; Enzyme assay; Enzymology
  13. Methods Mol Biol. 2023 ;2561 245-259
      Recent technical advances in mass spectrometry, as applied to the analytical chemistry of lipid molecules, enable the simultaneous detection of the multiplicity of lipid complex species present in the human brain. This, in combination with quantitative studies carried out in plasma samples, helps to identify disease biomarkers including for Alzheimer's disease (AD). Mass spectrometry imaging (MSI) is particularly powerful for the anatomical localization of lipids in brain slices, identifying lipid modifications in postmortem frozen samples from AD patients.Human brain tissues are sectioned in a cryostat and then covered with a chemical matrix, such as mercaptobenzothiazole (MBT) or α-cyano-4-hydroxycinnamic acid (CHCA), to ionize the lipid molecules either by sublimation or by spraying. We describe the use of matrix-assisted laser desorption ionization (MALDI) in an LTQ-Orbitrap-XL mass spectrometer to scan brain tissue slices with high spatial resolution, analyzing 50 μm cell layers. The lipid spectra obtained for each pixel are transformed to color-coded intensity maps of hundreds of lipid species included those within a single tissue slice.
    Keywords:  Alzheimer’s disease; Biomarkers; Lipid; MALDI-MSI; Mass spectroscopy; Neurolipid; UHPLC-MS
  14. Semin Cancer Biol. 2022 Oct 28. pii: S1044-579X(22)00209-7. [Epub ahead of print]87 32-47
      Cancer cells are characterized by sustained proliferation, which requires a huge demand of fuels to support energy production and biosynthesis. Energy is produced by the oxidation of the fuels during catabolism, and biosynthesis is achieved by the reduction of smaller units or precursors. Therefore, the oxidation-reduction (redox) reactions in cancer cells are more active compared to those in the normal counterparts. The higher activity of redox metabolism also induces a more severe oxidative stress, raising the question of how cancer cells maintain the redox balance. In this review, we overview the redox metabolism of cancer cells in an electron-tracing view. The electrons are derived from the nutrients in the tumor microenvironment and released during catabolism. Most of the electrons are transferred to NAD(P) system and then directed to four destinations: energy production, ROS generation, reductive biosynthesis and antioxidant system. The appropriate distribution of these electrons achieved by the function of redox regulation network is essential to maintain redox homeostasis in cancer cells. Interfering with the electron distribution and disrupting redox balance by targeting the redox regulation network may provide therapeutic implications for cancer treatment.
    Keywords:  Antioxidant system; Cancer metabolism; NADPH; ROS; Redox balance
  15. J Pharm Biomed Anal. 2022 Nov 09. pii: S0731-7085(22)00572-6. [Epub ahead of print]223 115151
      3-Hydroxyfatty acids (3-OH-FAs) are formed in the hydration step during mitochondrial β-oxidation of saturated straight-chain fatty acids, which is a catabolic pathway that involves several enzymes. For an unbiased biological interpretation, an enantioselective analysis of 3-OH-FAs including their stereoisomers is necessary, which may contribute to the elucidation of enzymatic mechanisms in the biological pathways. In this work, an enantioselective gradient UHPLC-MS/MS method based on 1.6 µm particle polysaccharide column (Chiralpak IA-U) for chiral separation of 3-hydroxyfatty acids was developed which covers carbon chain length from C8 to C18 with a good resolution of R and S enantiomers. The method is fast and sensitive for detecting enantiomers of 3-OH-FAs by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. A matrix matched-calibration strategy was applied for quantification of individual 3-OH-FA enantiomers. The method allows the simultaneous quantification of each enantiomer of 3-OH-FAs from C8-C18. One-phase liquid extraction with 2-propanol showed good extraction recoveries with over 90% on average. Further, the validated method was applied to investigate the alteration of 3-OH-FA enantiomers in platelets and plasma samples from human donors with different diagnoses of cardiovascular disease (acute coronary syndrome ACS, chronic coronary syndrome CCS). Both R and S enantiomers were detected in platelets and plasma samples with different predominance for R or S in dependence on carbon chain length, which might be associated with different functional enzymes of mitochondrial and peroxisomal β-oxidation. Finally, our study provides a new strategy for chiral separation and enantioselective analysis, showing great potential for targeted metabolomics in clinical biomarker discovery.
    Keywords:  Coronary artery disease; Enantioselective metabolomics; Enzyme steroselectivity; Mitochondrial fatty acid oxidation; Peroxysomal fatty acid oxidation; Targeted lipidomics
  16. Mol Cell. 2022 Nov 17. pii: S1097-2765(22)01055-3. [Epub ahead of print]82(22): 4246-4261.e11
      Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes via transfer of its acetyl group to proteins and metabolites. However, the synthesis and usage of acetyl-CoA in disease states such as cancer are poorly characterized. Here, we investigated global acetyl-CoA synthesis and protein acetylation in a mouse model and patient samples of hepatocellular carcinoma (HCC). Unexpectedly, we found that acetyl-CoA levels are decreased in HCC due to transcriptional downregulation of all six acetyl-CoA biosynthesis pathways. This led to hypo-acetylation specifically of non-histone proteins, including many enzymes in metabolic pathways. Importantly, repression of acetyl-CoA synthesis promoted oncogenic dedifferentiation and proliferation. Mechanistically, acetyl-CoA synthesis was repressed by the transcription factors TEAD2 and E2A, previously unknown to control acetyl-CoA synthesis. Knockdown of TEAD2 and E2A restored acetyl-CoA levels and inhibited tumor growth. Our findings causally link transcriptional reprogramming of acetyl-CoA metabolism, dedifferentiation, and cancer.
    Keywords:  E2A; HCC; TEAD2; acetyl-CoA metabolism; dedifferentiation; hepatocellular carcinoma; protein acetylation; transcriptional reprogramming
  17. Methods Mol Biol. 2023 ;2596 19-38
      The combination of large-scale protein separation techniques, sophisticated mass spectrometry, and systems bioinformatics has led to the establishment of proteomics as a distinct discipline within the wider field of protein biochemistry. Both discovery proteomics and targeted proteomics are widely used in biological and biomedical research, whereby the analytical approaches can be broadly divided into proteoform-centric top-down proteomics versus peptide-centric bottom-up proteomics. This chapter outlines the scientific value of top-down proteomics and describes how fluorescence two-dimensional difference gel electrophoresis can be combined with the systematic analysis of crucial post-translational modifications. The concept of on-membrane digestion following the electrophoretic transfer of proteins and the usefulness of comparative two-dimensional immunoblotting are discussed.
    Keywords:  Comparative proteomics; DIGE; Difference gel electrophoresis; Mass spectrometry; Top-down proteomics; Two-dimensional gel electrophoresis
  18. Nat Metab. 2022 Nov 14.
      The tumour microenvironment possesses mechanisms that suppress anti-tumour immunity. Itaconate is a metabolite produced from the Krebs cycle intermediate cis-aconitate by the activity of immune-responsive gene 1 (IRG1). While it is known to be immune modulatory, the role of itaconate in anti-tumour immunity is unclear. Here, we demonstrate that myeloid-derived suppressor cells (MDSCs) secrete itaconate that can be taken up by CD8+ T cells and suppress their proliferation, cytokine production and cytolytic activity. Metabolite profiling, stable-isotope tracing and metabolite supplementation studies indicated that itaconate suppressed the biosynthesis of aspartate and serine/glycine in CD8+ T cells to attenuate their proliferation and function. Host deletion of Irg1 in female mice bearing allografted tumours resulted in decreased tumour growth, inhibited the immune-suppressive activities of MDSCs, promoted anti-tumour immunity of CD8+ T cells and enhanced the anti-tumour activity of anti-PD-1 antibody treatment. Furthermore, we found a significant negative correlation between IRG1 expression and response to PD-1 immune checkpoint blockade in patients with melanoma. Our findings not only reveal a previously unknown role of itaconate as an immune checkpoint metabolite secreted from MDSCs to suppress CD8+ T cells, but also establish IRG1 as a myeloid-selective target in immunometabolism whose inhibition promotes anti-tumour immunity and enhances the efficacy of immune checkpoint protein blockade.
  19. Methods Mol Biol. 2023 ;2596 231-244
      Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms. Thus, 2D-DIGE analysis provides highly complementary data to non-gel-based shotgun mass spectrometry (MS) methods (e.g., liquid chromatography (LC)-MS/MS)-allowing, for example, identification of novel protein biomarkers for HCC or increasing insights into the molecular mechanisms underlying hepatocarcinogenesis.
    Keywords:  Comparative proteomics; Hepatocellular carcinoma (HCC); Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS); Liver cancer; Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS/MS); Mouse tissue; Two-dimensional difference gel electrophoresis (2D-DIGE)
  20. Biochem Biophys Res Commun. 2022 Nov 11. pii: S0006-291X(22)01562-5. [Epub ahead of print]637 144-152
      Cancer cells exhibit increased glutamine consumption compared to normal cells, supporting cell survival and proliferation. Glutamine is converted to α-ketoglutarate (αKG), which then enters the tricarboxylic acid cycle to generate ATP. Recently, therapeutic modulation of glutamine metabolism has become an attractive metabolic anti-cancer strategy. However, how synergistic combination therapy is required to overcome glutamine metabolism drug resistance remains elusive. To address this issue, we first investigated the role of αKG in regulating gene expression in several cancer cell lines. Using RNA-seq analysis and histone modification screening, we demonstrated that αKG reduced the expression of the immediate early gene (IEG) in cancer cells in an H3K27 acetylation-dependent manner. Conversely, glutaminase (GLS) inhibitors induce IEG expression in cancer cells. Furthermore, we showed that siRNA knockdown of orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) induces IEG expression. Notably, the NR4A1 agonist cytosporone B sensitizes GLS inhibitor resistance to cancer cell death. Together, these findings indicate that therapeutic targeting of IEG dysregulation by αKG can be a potentially effective anti-cancer therapeutic strategy for glutamine metabolism inhibitors.
    Keywords:  Cancer cells; GLS inhibitor; IEG; NR4A; αKG
  21. Metab Eng. 2022 Nov 09. pii: S1096-7176(22)00138-0. [Epub ahead of print]75 12-18
      Lipid biosynthesis plays a vital role in living cells and has been increasingly engineered to overproduce various lipid-based chemicals. However, owing to the tightly constrained and interconnected nature of lipid biosynthesis, both understanding and engineering of lipid metabolism remain challenging, even with the help of mathematical models. Here we report the development of a kinetic metabolic model of lipid metabolism in Saccharomyces cerevisiae that integrates fatty acid biosynthesis, glycerophospholipid metabolism, sphingolipid metabolism, storage lipids, lumped sterol synthesis, and the synthesis and transport of relevant target-chemicals, such as fatty acids and fatty alcohols. The model was trained on lipidomic data of a reference S. cerevisiae strain, single knockout mutants, and lipid overproduction strains reported in literature. The model was used to design mutants for fatty alcohol overproduction and the lipidomic analysis of the resultant mutant strains coupled with model-guided hypothesis led to discovery of a futile cycle in the triacylglycerol biosynthesis pathway. In addition, the model was used to explain successful and unsuccessful mutant designs in metabolic engineering literature. Thus, this kinetic model of lipid metabolism can not only enable the discovery of new phenomenon in lipid metabolism but also the engineering of mutant strains for overproduction of lipids.
    Keywords:  Fatty alcohol; Free fatty acid; Kinetic model; Lipid metabolism
  22. Front Microbiol. 2022 ;13 981994
      Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UPHLC-HRMS) is used to discover and monitor single or sets of biomarkers informing about metabolic processes of interest. The technique can detect 1000's of molecules (i.e., metabolites) in a single instrument run and provide a measurement of the global metabolome, which could be a fingerprint of activity. Despite the power of this approach, technical challenges have hindered the effective use of metabolomics to interrogate microbial communities implicated in the removal of priority contaminants. Herein, our efforts to circumvent these challenges and apply this emerging systems biology technique to microbiomes relevant for contaminant biodegradation will be discussed. Chlorinated ethenes impact many contaminated sites, and detoxification can be achieved by organohalide-respiring bacteria, a process currently assessed by quantitative gene-centric tools (e.g., quantitative PCR). This laboratory study monitored the metabolome of the SDC-9™ bioaugmentation consortium during cis-1,2-dichloroethene (cDCE) conversion to vinyl chloride (VC) and nontoxic ethene. Untargeted metabolomics using an UHPLC-Orbitrap mass spectrometer and performed on SDC-9™ cultures at different stages of the reductive dechlorination process detected ~10,000 spectral features per sample arising from water-soluble molecules with both known and unknown structures. Multivariate statistical techniques including partial least squares-discriminate analysis (PLSDA) identified patterns of measurable spectral features (peak patterns) that correlated with dechlorination (in)activity, and ANOVA analyses identified 18 potential biomarkers for this process. Statistical clustering of samples with these 18 features identified dechlorination activity more reliably than clustering of samples based only on chlorinated ethene concentration and Dhc 16S rRNA gene abundance data, highlighting the potential value of metabolomic workflows as an innovative site assessment and bioremediation monitoring tool.
    Keywords:  Dehalococcoidia; biomarkers; bioremediation; consortium SDC-9™; environmental monitoring; metabolomics; reductive dechlorination
  23. Ann Transl Med. 2022 Oct;10(20): 1115
      Background: Globally, the incidence and mortality of colorectal cancer (CRC) rank amongst the highest of all malignancies. Thus, research aimed at developing new screening strategies and biomarkers for the early detection of CRC is needed. At present, conventional screening methods have limitations; therefore, new testing strategies have been considered. Using metabolomics to explore the molecular changes in CRC tissue is a mainstream method for identifying potential biomarkers and key cancer factors.Methods: In the present study, 27 samples from nine CRC patients were used to analyze the metabolite differences between the tumor, paracancerous, and normal tissues. The metabolite differences in the various stages of CRC (stages IIA, IIB, and IIIC) were analyzed as well. Subsequently, principal component analysis (PCA), permutation, and trend analyses were performed. Weighted gene co-expression and metabolite-metabolite interaction networks were also constructed.
    Results: A total of 5,834 metabolites were identified among the included samples. Permutation analysis showed a clear separation between the different tissues and different stages. Compared with normal tissues, tumor tissues exhibited 11, 233, and 25 up-regulated metabolites as well as one, 77, and zero down-regulated metabolites in stages IIA, IIB, and IIIC, respectively. Moreover, tumor tissues in stage IIB exhibited more differential metabolites (233 up-regulated and 77 down-regulated). Weighted Gene Correlation Network Analysis (WGCNA) clustered the 5,834 metabolites into 15 different modules, of which four modules were significantly correlated with tissue specificity. Notably, glycerophospholipid metabolism, fatty acid metabolism, and other pathways were enriched in these modules.
    Conclusions: Fatty acids and glycerophospholipids were significantly related to the development of CRC. This result is of great significance for future targeted screening of CRC biomarkers and further clarifying the nutrient metabolism of cancer cells.
    Keywords:  Colorectal cancer (CRC); biomarker; fatty acids; metabolomics
  24. Methods Mol Biol. 2023 ;2596 291-302
      The biochemical and cell biological profiling of contractile fiber types and subcellular structures plays a central role in basic and applied myology. Mass spectrometry-based proteomics presents an ideal approach for the systematic identification of proteomic and subproteomic markers. These representative components of fast versus slow muscle fibers and their subcellular fractions are highly useful for in-depth surveys of skeletal muscle adaptations to physiological challenges, as well as the improvement of diagnostic, prognostic, and therapy-monitoring methodologies in muscle pathology. This chapter outlines the identification of subproteomic markers for skeletal muscle profiling based on bottom-up and top-down approaches, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE).
    Keywords:  DIGE; Proteomics; Skeletal muscle; Subcellular marker; Subproteomics
  25. Methods Mol Biol. 2023 ;2596 147-167
      Many biomedically relevant biomarkers are proteins with characteristic biochemical properties and a relatively restricted subcellular distribution. The comparative and mass spectrometry-based proteomic analysis of body fluids can be particularly instrumental for the targeted identification of novel protein biomarkers with pathological relevance. In this respect, new research efforts in biomarker discovery focus on the systematic mapping of the human saliva proteome, as well as the pathobiochemical identification of disease-related modifications or concentration changes in specific saliva proteins. As a product of exocrine secretion, saliva can be considered an ideal source for the biochemical identification of new disease indicators. Importantly, saliva represents a body fluid that is continuously available for diagnostic and prognostic assessments. This chapter gives an overview of saliva proteomics, including a discussion of the usefulness of both liquid chromatography and two-dimensional gel electrophoresis for efficient protein separation in saliva proteomics.
    Keywords:  Biofluid; Biomarker; DIGE; Diagnostics; Proteomics; Saliva
  26. Chin Med J (Engl). 2022 Nov 17.
      ABSTRACT: Tumor-associated macrophages (TAMs) are an essential proportion of tumor-infiltrating immune cells in the tumor microenvironment (TME) and have immunosuppressive functions. The high plasticity and corresponding phenotypic transformation of TAMs facilitate oncogenesis and progression, and suppress antineoplastic responses. Due to the uncontrolled proliferation of tumor cells, metabolism homeostasis is regulated, leading to a series of alterations in the metabolite profiles in the TME, which have a commensurate influence on immune cells. Metabolic reprogramming of the TME has a profound impact on the polarization and function of TAMs, and can alter their metabolic profiles. TAMs undergo a series of metabolic reprogramming processes, involving glucose, lipid, and amino acid metabolism, and other metabolic pathways, which terminally promote the development of the immunosuppressive phenotype. TAMs express a pro-tumor phenotype by increasing glycolysis, fatty acid oxidation, cholesterol efflux, and arginine, tryptophan, glutamate, and glutamine metabolism. Previous studies on the metabolism of TAMs demonstrated that metabolic reprogramming has intimate crosstalk with anti-tumor or pro-tumor phenotypes and is crucial for the function of TAMs themselves. Targeting metabolism-related pathways is emerging as a promising therapeutic modality because of the massive metabolic remodeling that occurs in malignant cells and TAMs. Evidence reveals that the efficacy of immune checkpoint inhibitors is improved when combined with therapeutic strategies targeting metabolism-related pathways. In-depth research on metabolic reprogramming and potential therapeutic targets provides more options for anti-tumor treatment and creates new directions for the development of new immunotherapy methods. In this review, we elucidate the metabolic reprogramming of TAMs and explore how they sustain immunosuppressive phenotypes to provide a perspective for potential metabolic therapies.
  27. Methods Mol Biol. 2023 ;2596 217-230
      Several years have passed since LC (liquid chromatography)-MS (mass spectrometry) became the mainstream for proteomic analysis; however, conventional fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) continues to be an important technology that enables rapid and direct visualization of hundreds to thousands of proteins and their quantitative analyses. We can get global proteomic views using 2D-DIGE within 3 days and then identify proteins with differential expression levels using MALDI-TOF/MS and MASCOT search engine. Here, we describe our routine 2D-DIGE proteomic analysis of the liver isolated from mice in pathological conditions within 1 week.
    Keywords:  2D-DIGE; Isoelectric focusing; MALDI-TOF/MS; Proteomics
  28. Cell Biosci. 2022 Nov 12. 12(1): 183
      BACKGROUND: Exploiting cancer metabolism during nutrient availability holds immense potential for the clinical and therapeutic benefits of hepatocellular carcinoma (HCC) patients. Dietary methionine is a metabolic dependence of cancer development, but how the signal transduction integrates methionine status to achieve the physiological demand of cancer cells remains unknown.METHODS: Low or high levels of dietary methionine was fed to mouse models with patient-derived xenograft or diethyl-nitrosamine induced liver cancer. RNA sequence and metabolomics were performed to reveal the profound effect of methionine restriction on gene expression and metabolite changes. Immunostaining, sphere formation assays, in vivo tumourigenicity, migration and self-renewal ability were conducted to demonstrate the efficacy of methionine restriction and sorafenib.
    RESULTS: We discovered that mTORC1-c-Myc-SIRT4 axis was abnormally regulated in a methionine-dependent manner and affected the HCC progression. c-Myc rewires methionine metabolism through TRIM32 mediated degradation of SIRT4, which regulates MAT2A activity by ADP-ribosylation on amino acid residue glutamic acid 111. MAT2A is a key enzyme to generate S-adenosylmethionine (SAM). Loss of SIRT4 activates MAT2A, thereby increasing SAM level and dynamically regulating gene expression, which triggers the high proliferation rate of tumour cells. SIRT4 exerts its tumour suppressive function with targeted therapy (sorafenib) by affecting methionine, redox and nucleotide metabolism.
    CONCLUSIONS: These findings establish a novel characterization of the signaling transduction and the metabolic consequences of dietary methionine restriction in malignant liver tissue of mice. mTORC1, c-Myc, SIRT4 and ADP ribosylation site of MAT2A are promising clinical and therapeutic targets for the HCC treatment.
    Keywords:  ADP ribosylation; Cancer; MAT2A; Methionine metabolism; SIRT4; TRIM32; c-Myc; mTOR
  29. J Chromatogr A. 2022 Nov 03. pii: S0021-9673(22)00793-2. [Epub ahead of print]1685 463602
      Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis. Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD), metabolic disorders, and cancer. It is consequently critical to develop a reliable, quantitative method for the analysis of tryptophan and its downstream metabolites from the kynurenine, serotonin, and indoles pathways. An LC-MS/MS method was designed for the analysis of tryptophan and 20 of its metabolites, without derivatization and performed in a single run. This method was validated for both serum and stool. The comparisons between serum and plasma, collected with several differing anticoagulants, showed significant differences only for serotonin. References values were established in sera and stools from healthy donors. For stool samples, as a proof of concept, the developed method was applied to a healthy control group and an IBD patient group. Results showed significant differences in the concentrations of tryptophan, xanthurenic acid, kynurenic acid, indole-3-lactic acid, and picolinic acid. This method allowed an extensive analysis of the three tryptophan metabolic pathways in two compartments. Beyond the application to IBD patients, the clinical use of this method is wide-ranging and may be applied to other pathological conditions involving tryptophan metabolism, such as neurological, psychiatric, or auto-inflammatory pathologies.
    Keywords:  Inflammatory bowel diseases; LC-MS/MS; tryptophan metabolites profile
  30. Mol Omics. 2022 Nov 14.
      Metabolomics, the large-scale study of metabolites, has significant appeal as a source of information for metabolic modeling and other scientific applications. One common approach for measuring metabolomics data is gas chromatography-mass spectrometry (GC-MS). However, GC-MS metabolomics data are typically reported as relative abundances, precluding their use with approaches and tools where absolute concentrations are necessary. While chemical standards can be used to help provide quantification, their use is time-consuming, expensive, or even impossible due to their limited availability. The ability to infer absolute concentrations from GC-MS metabolomics data without chemical standards would have significant value. We hypothesized that when analyzing time-course metabolomics datasets, the mass balances of metabolism and other biological information could provide sufficient information towards inference of absolute concentrations. To demonstrate this, we developed and characterized MetaboPAC, a computational framework that uses two approaches-one based on kinetic equations and another using biological heuristics-to predict the most likely response factors that allow translation between relative abundances and absolute concentrations. When used to analyze noiseless synthetic data generated from multiple types of kinetic rate laws, MetaboPAC performs significantly better than negative control approaches when 20% of kinetic terms are known a priori. Under conditions of lower sampling frequency and high noise, MetaboPAC is still able to provide significant inference of concentrations in 3 of 4 models studied. This provides a starting point for leveraging biological knowledge to extract concentration information from time-course intracellular GC-MS metabolomics datasets, particularly for systems that are well-studied and have partially known kinetic structures.
  31. Exp Cell Res. 2022 Nov 15. pii: S0014-4827(22)00420-7. [Epub ahead of print] 113427
      Protein kinase C epsilon (PKCε) belongs to a family of serine/threonine kinases that control cell proliferation, differentiation and survival. Aberrant PKCε activation and overexpression is a frequent feature of numerous cancers. However, its role in regulation of lipid metabolism in cancer cells remains elusive. Here we report a novel function of PKCε in regulating of prostate cancer cell proliferation by modulation of PKM2-mediated de novo lipogenesis. We show that PKCε promotes de novo lipogenesis and tumor cell proliferation via upregulation of lipogenic enzymes and lipid contents in prostate cancer cells. Mechanistically, PKCε interacts with NABD (1-388) domain of C-terminal deletion on pyruvate kinase isoform M2 (PKM2) and enhances the Tyr105 phosphorylation of PKM2, leading to its nuclear localization. Moreover, forced expression of mutant Tyr105 (Y105F) or PKM2 inhibition suppressed de novo lipogenesis and cell proliferation induced by overexpression of PKCε in prostate cancer cells. In a murine tumor model, inhibitor of PKM2 antagonizes lipogenic enzymes expression and prostate cancer growth induced by overexpression of PKCε in vivo. These data indicate that PKCε is a critical regulator of de novo lipogenesis, which may represent a potential therapeutic target for the treatment of prostate cancer.
    Keywords:  De novo lipogenesis; PKCε; PKM2; Prostate cancer; Tumor growth
  32. Curr Med Chem. 2022 Nov 11.
      Lipid metabolism is a complex biochemical process that regulates normal cell activity and death. Ferroptosis is a novel mode of programmed cell death different from apoptosis, pyroptosis, and autophagy. Abnormal lipid metabolism may lead to lipid peroxidation and cell rupture death, which are regulated by lipoxygenase(LOX), long-chain acyl-coA synthases, and antioxidant enzymes. Alternatively, Fe2+ and Fe3+ are required for the activity of LOXs and ferroptosis, and Fe2+ can significantly accelerate lipid peroxidation in ferroptosis. Abnormal lipid metabolism is a certain risk factor for cardiovascular disease. In recent years, the important role of ferroptosis in developing cardiovascular disease has been increasingly reported. Reducing lipid accumulation could reduce the occurrence of ferroptosis, thus alleviating cardiovascular disease deterioration. This article reviews the relationship of lipid peroxidation to the general mechanism of ferroptosis and highlights lipid peroxidation as the common point of ferroptosis and cardiovascular disease.
    Keywords:  cardiovascular disease; ferroptosis; lipid metabolism; mechanism
  33. Biomed Pharmacother. 2022 Nov 14. pii: S0753-3322(22)01381-6. [Epub ahead of print]157 113992
      Abnormal intracellular metabolism not only provides nutrition for tumor occurrence and development, but also sensitizes the function of various immune cells in the immune microenvironment to promote tumor immune escape. This review discusses the emerging role of immune cells in the progress of pancreatic cancer, acrossing metabolic reprogramming and key metabolic pathways present in different immune cell types. At present, the hotspots of metabolic reprogramming of immune cells in pancreatic cancer progression mainly focuses on glucose metabolism, lipid metabolism, tricarboxylic acid cycle and amino acid metabolism, which affect the function of anti-tumor immune cells and immunosuppressive cells in the microenvironment, such as macrophages, dendritic cells, T cells, myeloid-derived suppressor cells, neutrophils and B cells by a series of key metabolic signaling pathways, such as PI3K/AKT, mTOR, AMPK, HIF-1α, c-Myc and p53. Drugs that target the tumor metabolism pathways for clinical treatment of pancreatic cancer are also systematically elaborated, which may constitute food for others' projects involved in clinical anti-cancer research.
    Keywords:  Immune cells; Immunotherapy; Metabolic reprogramming; Pancreatic cancer
  34. Acta Neuropathol Commun. 2022 Nov 14. 10(1): 165
      Lipid peroxidation is a process of oxidative degradation of cellular lipids that is increasingly recognized as an important factor in the pathogenesis of neurodegenerative diseases. We were therefore interested in the manifestation of lipid peroxidation in synucleinopathies, a group of neurodegenerative diseases characterized by the central pathology of α-synuclein aggregates, including Parkinson's disease, multiple system atrophy, dementia with Lewy bodies and Alzheimer's disease with Lewy bodies. We assessed lipid peroxidation products, lipid aldehydes, in the amygdala, a common disease-affected region in synucleinopathies, and in the visual cortex, a disease-unaffected region. We found that the levels of lipid aldehydes were significantly increased in the amygdala, but not in the visual cortex. We hypothesized that these increases are due to increases in the abundance of unsaturated lipids, since lipid aldehydes are formed from unsaturated lipids. We undertook a comprehensive analysis of membrane lipids using liquid chromatography-mass spectrometry and found that unsaturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were specifically elevated in the amygdala and correlated with increases in lipid aldehydes. Furthermore, unsaturated phosphatidylethanolamine levels were associated with soluble α-synuclein. Put together, these results suggest that manifestation of lipid peroxidation is prevalent in synucleinopathies and is likely to be due to increases in unsaturated membrane lipids. Our findings underscore the importance of lipid peroxidation in α-synuclein pathology and in membrane structure maintenance.
    Keywords:  Lipid aldehydes; Lipid peroxidation; Parkinson’s disease; Synucleinopathies; Unsaturated lipids