bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2023‒01‒15
twenty-six papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. J Proteome Res. 2023 Jan 09.
      Liquid chromatography coupled with bottom-up mass spectrometry (LC-MS/MS)-based proteomics is a versatile technology for identifying and quantifying proteins in complex biological mixtures. Postidentification, analysis of changes in protein abundances between conditions requires increasingly complex and specialized statistical methods. Many of these methods, in particular the family of open-source Bioconductor packages MSstats, are implemented in a coding language such as R. To make the methods in MSstats accessible to users with limited programming and statistical background, we have created MSstatsShiny, an R-Shiny graphical user interface (GUI) integrated with MSstats, MSstatsTMT, and MSstatsPTM. The GUI provides a point and click analysis pipeline applicable to a wide variety of proteomics experimental types, including label-free data-dependent acquisitions (DDAs) or data-independent acquisitions (DIAs), or tandem mass tag (TMT)-based TMT-DDAs, answering questions such as relative changes in the abundance of peptides, proteins, or post-translational modifications (PTMs). To support reproducible research, the application saves user's selections and builds an R script that programmatically recreates the analysis. MSstatsShiny can be installed locally via Github and Bioconductor, or utilized on the cloud at We illustrate the utility of the platform using two experimental data sets (MassIVE IDs MSV000086623 and MSV000085565).
    Keywords:  bioinformatics; differential analysis; graphical user interface; mass spectrometry; post-translational modifications; proteomics; software
  2. Eur J Pharmacol. 2023 Jan 06. pii: S0014-2999(23)00008-0. [Epub ahead of print]941 175497
      Cancer often perturbs lipid metabolism, which leads to the alteration of metabolism intermediates, contributing to their deregulated growth and metastasis. Alteration of lipid metabolism shifting to contain more polyunsaturated fatty acids (PUFAs) in membrane phospholipids (PLs) also leads to cancer therapy resistance. High amounts of PL-PUFAs render cancer cells more vulnerable to lipid peroxidation (LPO), predisposing them towards ferroptosis, a new form of iron-dependent oxidative regulated cell death. The commitment of cancer undergoing ferroptotic cell death depends on the adaptive lipidome remodeling, LPO patterns, and LPO scavenging ability in heterogeneous cancer cells. Ferroptosis is receiving attention in cancer research as treating cancers, altering membrane lipid homeostasis, and refractory from conventional therapies. Therefore, a better understanding of the molecular underpinning of lipid metabolism alterations may provide new opportunities for solving cancer resistance. This review intends to understand altered lipid metabolism in cancers and discuss lipid composition and metabolic processes associated with ferroptosis induction in cancers.
    Keywords:  Cancer; Ferroptosis; Lipid metabolism; Lipid peroxidation; Polyunsaturated fatty acids
  3. Nat Commun. 2023 Jan 06. 14(1): 94
      A plethora of software suites and multiple classes of spectral libraries have been developed to enhance the depth and robustness of data-independent acquisition (DIA) data processing. However, how the combination of a DIA software tool and a spectral library impacts the outcome of DIA proteomics and phosphoproteomics data analysis has been rarely investigated using benchmark data that mimics biological complexity. In this study, we create DIA benchmark data sets simulating the regulation of thousands of proteins in a complex background, which are collected on both an Orbitrap and a timsTOF instruments. We evaluate four commonly used software suites (DIA-NN, Spectronaut, MaxDIA and Skyline) combined with seven different spectral libraries in global proteome analysis. Moreover, we assess their performances in analyzing phosphopeptide standards and TNF-α-induced phosphoproteome regulation. Our study provides a practical guidance on how to construct a robust data analysis pipeline for different proteomics studies implementing the DIA technique.
  4. Foods. 2022 Dec 28. pii: 163. [Epub ahead of print]12(1):
      Milk is a rich source of lipids, with the major components being triglycerides (TAG) and phospholipids (mainly phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)). Liquid chromatography-mass spectrometry (LC-MS) is the predominant technique for lipid identification and quantification across all biological samples. While fatty acid (FA) composition of the major lipid classes of milk can be readily determined using tandem MS, elucidating the regio-distribution and double bond position of the FA remains difficult. Various workflows have been reported on the quantification of lipid species in biological samples in the past 20 years, but no standard or consensus methods are currently available for the quantification of milk phospholipids. This study will examine the influence of several common factors in lipid analysis workflow (including lipid extraction protocols, LC stationary phases, mobile phase buffers, gradient elution programmes, mass analyser resolution and isotope correction) on the quantification outcome of bovine milk phospholipids. The pros and cons of the current LC-MS methods as well as the critical problems to be solved will also be discussed.
    Keywords:  liquid chromatography-mass spectrometry; milk; phospholipids; quantification
  5. Int J Mol Sci. 2022 Dec 28. pii: 537. [Epub ahead of print]24(1):
      Primary liver cancer is a heterogeneous disease. Liver cancer metabolism includes both the reprogramming of intracellular metabolism to enable cancer cells to proliferate inappropriately and adapt to the tumor microenvironment and fluctuations in regular tissue metabolism. Currently, metabolomics and metabolite profiling in liver cirrhosis, liver cancer, and hepatocellular carcinoma (HCC) have been in the spotlight in terms of cancer diagnosis, monitoring, and therapy. Metabolomics is the global analysis of small molecules, chemicals, and metabolites. Metabolomics technologies can provide critical information about the liver cancer state. Here, we review how liver cirrhosis, liver cancer, and HCC therapies interact with metabolism at the cellular and systemic levels. An overview of liver metabolomics is provided, with a focus on currently available technologies and how they have been used in clinical and translational research. We also list scalable methods, including chemometrics, followed by pathway processing in liver cancer. We conclude that important drivers of metabolomics science and scientific technologies are novel therapeutic tools and liver cancer biomarker analysis.
    Keywords:  liver cancer; metabolites; metabolomics; microbiome; scientific technology
  6. Front Immunol. 2022 ;13 955476
      Cancer is a heterogeneous disease characterized by various genetic and phenotypic aberrations. Cancer cells undergo genetic modifications that promote their proliferation, survival, and dissemination as the disease progresses. The unabated proliferation of cancer cells incurs an enormous energy demand that is supplied by metabolic reprogramming. Cancer cells undergo metabolic alterations to provide for increased energy and metabolite requirement; these alterations also help drive the tumor progression. Dysregulation in glucose uptake and increased lactate production via "aerobic glycolysis" were described more than 100 years ago, and since then, the metabolic signature of various cancers has been extensively studied. However, the extensive research in this field has failed to translate into significant therapeutic intervention, except for treating childhood-ALL with amino acid metabolism inhibitor L-asparaginase. Despite the growing understanding of novel metabolic alterations in tumors, the therapeutic targeting of these tumor-specific dysregulations has largely been ineffective in clinical trials. This chapter discusses the major pathways involved in the metabolism of glucose, amino acids, and lipids and highlights the inter-twined nature of metabolic aberrations that promote tumorigenesis in different types of cancer. Finally, we summarise the therapeutic interventions which can be used as a combinational therapy to target metabolic dysregulations that are unique or common in blood, breast, colorectal, lung, and prostate cancer.
    Keywords:  cancer; cancer metabolism; cancer microenvironment; metabolic reprogramming; targeted therapy
  7. Expert Opin Drug Metab Toxicol. 2023 Jan 07.
      BACKGROUND: 4-hydroxy-N,N,methylpropyltryptamine (4-OH-MPT) is a psychedelic tryptamine whose use is regulated in several countries. Due to unspecific effects, consumption can be ascertained only through toxicological analyses. However, the trace amounts of tryptamines usually are challenging to detect in biological samples. 4-OH-MPT metabolism was characterized to identify optimal metabolite markers of intake in clinical/forensic toxicology.RESEARCH DESIGN AND METHODS: 4-OH-MPT was incubated with 10-donor-pooled human hepatocytes to simulate in vivo conditions; samples were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and data were processed with Compound Discoverer from Thermo Scientific. LC-HRMS/MS and data mining were supported by in silico metabolite predictions (GLORYx).
    RESULTS: Three phase I and 4 phase II metabolites were identified, including N-oxidation and N-demethylation at the alkylamine chain, and O-glucuronidation and sulfation at the hydroxylindole core.
    CONCLUSIONS: 4-OH-MPT metabolic fate was consistent with the human metabolism of tryptamine analogues: we suggest 4-OH-MPT-N-oxide and 4-hydroxy-N,N-propyltryptamine (4-OH-PT) as metabolite biomarkers of 4-OH-MPT consumption after glucuronide/sulfate hydrolysis in biological samples to improve the detection of 4-OH-MPT and phase I metabolites; 4-OH-MPT-glucuronide is suggested as an additional biomarker when hydrolysis is not performed. Further research on the metabolism of structural analogues is necessary to evaluate the specificity of 4-OH-MPT metabolite biomarkers.
    Keywords:  4-OH-MPT; Human hepatocyte metabolism; Liquid chromatography-high-resolution tandem mass spectrometry; Meprocin; Novel psychoactive substance; Software-assisted data mining; Tryptamine
  8. Int J Mol Sci. 2022 Dec 20. pii: 12. [Epub ahead of print]24(1):
      Despite the remarkable progress in cancer treatment up to now, we are still far from conquering the disease. The most substantial change after the malignant transformation of normal cells into cancer cells is the alteration in their metabolism. Cancer cells reprogram their metabolism to support the elevated energy demand as well as the acquisition and maintenance of their malignancy, even in nutrient-poor environments. The metabolic alterations, even under aerobic conditions, such as the upregulation of the glucose uptake and glycolysis (the Warburg effect), increase the ROS (reactive oxygen species) and glutamine dependence, which are the prominent features of cancer metabolism. Among these metabolic alterations, high glutamine dependency has attracted serious attention in the cancer research community. In addition, the oncogenic signaling pathways of the well-known important genetic mutations play important regulatory roles, either directly or indirectly, in the central carbon metabolism. The identification of the convergent metabolic phenotypes is crucial to the targeting of cancer cells. In this review, we investigate the relationship between cancer metabolism and the signal transduction pathways, and we highlight the recent developments in anti-cancer therapy that target metabolism.
    Keywords:  ROS; aerobic glycolysis; anti-cancer drug; cancer; glutamine; metabolism; redox; signal transduction
  9. Curr Protoc. 2023 Jan;3(1): e644
      Accurate identification and quantification of drugs and their metabolites (analytes) in biological matrices is an analytical foundation of clinical and forensic toxicology. For decades, liquid chromatography interfaced by electrospray ionization with tandem mass spectrometry (LC-ESI-MS/MS) has been a widely used technology for analysis in the field of toxicology, as well as in many other fields of bioscience. It is also known that ion response in LC-ESI-MS/MS analysis is influenced by coeluting biological compounds and that preanalytical sample clean-up is often insufficient in removing these interferences. As a result, a normalization technique is commonly used for assessment and compensation of matrix effects encountered in routine analysis. Internal standardization with a stable isotope analog of the analyte is the predominant normalization technique used in LC-ESI-MS/MS analysis. The technique, however, requires commercial availability or costly custom synthesis of an isotopic analog specific for each analyte. Here we describe an alternative technique for matrix normalization for use in high-volume, multianalyte testing without the need for isotope analogs. The technique involves analysis of the original sample (neat analysis) followed by analysis of a second sample aliquot (spike analysis) that has been fortified with a controlled amount of reference analyte. A calibration procedure similar to internal standardization is employed, using an ion response ratio of neat to fortified analyte. As a demonstration of the technique in multianalyte testing, we provide a detailed protocol for simultaneous detection and quantification of 102 drugs and drug metabolites in human urine. We also provide a support protocol for addition of new analytes to the multianalyte panel, allowing convenient collection of the validation data during routine testing. The matrix normalization technique and testing principles may be applicable to a wide range of analytes and biological matrices, not only those encountered in toxicology but also in other fields of bioscience. © 2023 Wiley Periodicals LLC. Basic Protocol: Detection and quantification of 102 toxicology analytes in urine by LC-ESI-MS/MS analysis using the threshold accurate calibration technique Support Protocol: Method for addition and validation of new analytes to expand the Basic Protocol.
    Keywords:  TAC; electrospray ionization; liquid chromatography; matrix effect normalization; tandem mass spectrometry; threshold accurate calibration
  10. Molecules. 2022 Dec 30. pii: 312. [Epub ahead of print]28(1):
      The current approach to equine anti-doping is focused on the targeted detection of prohibited substances. However, as new substances are rapidly being developed, the need for complimentary methods for monitoring is crucial to ensure the integrity of the racing industry is upheld. Lipidomics is a growing field involved in the characterisation of lipids, their function and metabolism in a biological system. Different lipids have various biological effects throughout the equine system including platelet aggregation and inflammation. A certain class of lipids that are being reviewed are the eicosanoids (inflammatory markers). The use of eicosanoids as a complementary method for monitoring has become increasingly popular with various studies completed to highlight their potential. Studies including various corticosteroids, non-steroidal anti-inflammatories and cannabidiol have been reviewed to highlight the progress lipidomics has had in contributing to the equine anti-doping industry. This review has explored the techniques used to prepare and analyse samples for lipidomic investigations in addition to the statistical analysis and potential for lipidomics to be used for a longitudinal assessment in the equine anti-doping industry.
    Keywords:  NSAIDs; analytical; corticosteroids; lipidomics; review
  11. Adv Sci (Weinh). 2023 Jan 10. e2204006
      Emerging evidence reveals that amino acid metabolism plays an important role in ferroptotic cell death. The conversion of methionine to cysteine is well known to protect tumour cells from ferroptosis upon cysteine starvation through transamination. However, whether amino acids-produced metabolites participate in ferroptosis independent of the cysteine pathway is largely unknown. Here, the authors show that the tryptophan metabolites serotonin (5-HT) and 3-hydroxyanthranilic acid (3-HA) remarkably facilitate tumour cells to escape from ferroptosis distinct from cysteine-mediated ferroptosis inhibition. Mechanistically, both 5-HT and 3-HA act as potent radical trapping antioxidants (RTA) to eliminate lipid peroxidation, thereby inhibiting ferroptotic cell death. Monoamine oxidase A (MAOA) markedly abrogates the protective effect of 5-HT via degrading 5-HT. Deficiency of MAOA renders cancer cells resistant to ferroptosis upon 5-HT treatment. Kynureninase (KYNU), which is essential for 3-HA production, confers cells resistant to ferroptotic cell death, whereas 3-hydroxyanthranilate 3,4-dioxygenase (HAAO) significantly blocks 3-HA mediated ferroptosis inhibition by consuming 3-HA. In addition, the expression level of HAAO is positively correlated with lipid peroxidation and clinical outcome. Together, the findings demonstrate that tryptophan metabolism works as a new anti-ferroptotic pathway to promote tumour growth, and targeting this pathway will be a promising therapeutic approach for cancer treatment.
    Keywords:  3-HA; 3-hydroxyanthranilate 3,4-dioxygenase (HAAO); 5-HT; ferroptosis; kynureninase (KYNU; monoamine oxidase A (MAOA) ; tryptophan
  12. Cancers (Basel). 2022 Dec 22. pii: 61. [Epub ahead of print]15(1):
      Despite extensive research, the 5-year survival rate of pancreatic cancer (PDAC) patients remains at only 9%. Patients often show poor treatment response, due partly to a highly complex tumor microenvironment (TME). Cancer-associated fibroblast (CAF) heterogeneity is characteristic of the pancreatic TME, where several CAF subpopulations have been identified, such as myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen presenting CAFs (apCAFs). In PDAC, cancer cells continuously adapt their metabolism (metabolic switch) to environmental changes in pH, oxygenation, and nutrient availability. Recent advances show that these environmental alterations are all heavily driven by stromal CAFs. CAFs and cancer cells exchange cytokines and metabolites, engaging in a tight bidirectional crosstalk, which promotes tumor aggressiveness and allows constant adaptation to external stress, such as chemotherapy. In this review, we summarize CAF diversity and CAF-mediated metabolic rewiring, in a PDAC-specific context. First, we recapitulate the most recently identified CAF subtypes, focusing on the cell of origin, activation mechanism, species-dependent markers, and functions. Next, we describe in detail the metabolic crosstalk between CAFs and tumor cells. Additionally, we elucidate how CAF-driven paracrine signaling, desmoplasia, and acidosis orchestrate cancer cell metabolism. Finally, we highlight how the CAF/cancer cell crosstalk could pave the way for new therapeutic strategies.
    Keywords:  CAF; PDAC; acidosis; cancer-associated fibroblast; desmoplasia; hypoxia; metabolism; pancreatic cancer; paracrine signaling
  13. Asian J Androl. 2023 Jan 10.
      Reprogramming of metabolism is a hallmark of tumors, which has been explored for therapeutic purposes. Prostate cancer (PCa), particularly advanced and therapy-resistant PCa, displays unique metabolic properties. Targeting metabolic vulnerabilities in PCa may benefit patients who have exhausted currently available treatment options and improve clinical outcomes. Among the many nutrients, glutamine has been shown to play a central role in the metabolic reprogramming of advanced PCa. In addition to amino acid metabolism, glutamine is also widely involved in the synthesis of other macromolecules and biomasses. Targeting glutamine metabolic network by maximally inhibiting glutamine utilization in tumor cells may significantly add to treatment options for many patients. This review summarizes the metabolic landscape of PCa, with a particular focus on recent studies of how glutamine metabolism alterations affect therapeutic resistance and disease progression of PCa, and suggests novel therapeutic strategies.
    Keywords:  castration resistance; glutamine metabolism; prostate cancer; tumor metabolism
  14. J Huntingtons Dis. 2023 Jan 04.
      BACKGROUND: Metabolic abnormalities have long been predicted in Huntington's disease (HD) but remain poorly characterized. Chronobiological dysregulation has been described in HD and may include abnormalities in circadian-driven metabolism.OBJECTIVE: Here we investigated metabolite profiles in the transgenic sheep model of HD (OVT73) at presymptomatic ages. Our goal was to understand changes to the metabolome as well as potential metabolite rhythm changes associated with HD.
    METHODS: We used targeted liquid chromatography mass spectrometry (LC-MS) metabolomics to analyze metabolites in plasma samples taken from female HD transgenic and normal (control) sheep aged 5 and 7 years. Samples were taken hourly across a 27-h period. The resulting dataset was investigated by machine learning and chronobiological analysis.
    RESULTS: The metabolic profiles of HD and control sheep were separable by machine learning at both ages. We found both absolute and rhythmic differences in metabolites in HD compared to control sheep at 5 years of age. An increase in both the number of disturbed metabolites and the magnitude of change of acrophase (the time at which the rhythms peak) was seen in samples from 7-year-old HD compared to control sheep. There were striking similarities between the dysregulated metabolites identified in HD sheep and human patients (notably of phosphatidylcholines, amino acids, urea, and threonine).
    CONCLUSION: This work provides the first integrated analysis of changes in metabolism and circadian rhythmicity of metabolites in a large animal model of presymptomatic HD.
    Keywords:  Huntington’s disease; chronobiology; circadian rhythms; machine learning; mass spectrometry; metabolomics
  15. J Mass Spectrom Adv Clin Lab. 2023 Jan;27 49-55
      In the pursuit of personalized diagnostics and tailored treatments, quantitative protein tests contribute to a more precise definition of health and disease. The development of new quantitative protein tests should be driven by an unmet clinical need and performed in a collaborative effort that involves all stakeholders. With regard to the analytical part, mass spectrometry (MS)-based platforms are an excellent tool for quantification of specific proteins in body fluids, for example focused on cancer. The obtained readouts have great potential in determining tumor aggressiveness to facilitate treatment decisions, and can furthermore be used to monitor patient response. Internationally standardized TNM classifications of malignant tumors are beneficial for diagnosis, however treatment outcome and survival of cancer patients is poorly predicted. To this end, the importance of the tumor microenvironment has endorsed the introduction of the tumor-stroma ratio as a prognostic parameter in solid primary tumor types. Currently, the stromal content of tumor tissues is determined via routine diagnostic pathology slides. With the development of liquid chromatography (LC)-MS methods we aim at quantification of tumor-stroma specific proteins in body fluids. In this mini-review the analytical aspect of this developmental trajectory is further detailed.
    Keywords:  Cancer proteomics; Clinical chemistry; Liquid biopsy; Protein quantification; Tumor-stroma ratio
  16. Int J Mol Sci. 2022 Dec 22. pii: 210. [Epub ahead of print]24(1):
      With the rising incidence of hepatocellular carcinoma (HCC) from non-alcoholic steatohepatitis (NASH), identifying new metabolic readouts that function in metabolic pathway perpetuation is still a demand. The study aimed to compare the metabolic signature between NASH and NASH-HCC patients to explore novel reprogrammed metabolic pathways that might modulate cancer progression in NASH patients. NASH and NASH-HCC patients were recruited and screened for metabolomics, and isotope-labeled lipidomics were targeted and profiled using the EXION-LCTM system equipped with a Triple-TOFTM 5600+ system. Results demonstrated significantly (p ≤ 0.05) higher levels of triacylglycerol, AFP, AST, and cancer antigen 19-9 in NASH-HCC than in NASH patients, while prothrombin time, platelet count, and total leukocyte count were decreased significantly (p ≤ 0.05). Serum metabolic profiling showed a panel of twenty metabolites with 10% FDR and p ≤ 0.05 in both targeted and non-targeted analysis that could segregate NASH-HCC from NASH patients. Pathway analysis revealed that the metabolites are implicated in the down-regulation of necroptosis, amino acid metabolism, and regulation of lipid metabolism by PPAR-α, biogenic amine synthesis, fatty acid metabolism, and the mTOR signaling pathway. Cholesterol metabolism, DNA repair, methylation pathway, bile acid, and salts metabolism were significantly upregulated in NASH-HCC compared to the NASH group. Metabolite-protein interactions network analysis clarified a set of well-known protein encoding genes that play crucial roles in cancer, including PEMT, IL4I1, BAAT, TAT, CDKAL1, NNMT, PNP, NOS1, and AHCYL. Taken together, reliable metabolite fingerprints are presented and illustrated in a detailed map for the most predominant reprogrammed metabolic pathways that target HCC development from NASH.
    Keywords:  hepatocellular carcinoma; lipidomic; metabolomic; non-alcoholic steatohepatitis; pathway reprogramming
  17. Nutrients. 2022 Dec 20. pii: 3. [Epub ahead of print]15(1):
      Tumour metabolomics and transcriptomics co-expression network as related to biological folate alteration and cancer malignancy remains unexplored in human non-small cell lung cancers (NSCLC). To probe the diagnostic biomarkers, tumour and pair lung tissue samples (n = 56) from 97 NSCLC patients were profiled for ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS)-analysed metabolomics, targeted transcriptionomics, and clinical folate traits. Weighted Gene Co-expression Network Analysis (WGCNA) was performed. Tumour lactate was identified as the top VIP marker to predict advance NSCLC (AUC = 0.765, Sig = 0.017, CI 0.58-0.95). Low folate (LF)-tumours vs. adjacent lungs displayed higher glycolytic index of lactate and glutamine-associated amino acids in enriched biological pathways of amino sugar and glutathione metabolism specific to advance NSCLCs. WGCNA classified the green module for hub serine-navigated glutamine metabolites inversely associated with tumour and RBC folate, which module metabolites co-expressed with a predominant up-regulation of LF-responsive metabolic genes in glucose transport (GLUT1), de no serine synthesis (PHGDH, PSPH, and PSAT1), folate cycle (SHMT1/2 and PCFR), and down-regulation in glutaminolysis (SLC1A5, SLC7A5, GLS, and GLUD1). The LF-responsive WGCNA markers predicted poor survival rates in lung cancer patients, which could aid in optimizing folate intervention for better prognosis of NSCLCs susceptible to folate malnutrition.
    Keywords:  WGCNA; non–small-cell lung cancers; target metabolomics; transcriptional profile; tumour folate
  18. Biomed Chromatogr. 2023 Jan 12. e5583
      Understanding the protein dynamics of a drug target is important for pharmaceutical research because it provides insight into drug design, target engagement, pharmacodynamics, and drug efficacy. Non-radioactive isotope labeling has been the method-of-choice for protein turnover measurement thanks to the advancement of high-resolution mass spectrometry (HRMS). While the changes in proteome in cell cultures can be monitored precisely, as the culture media can be completely replaced with 2 H-, 15 N- or 13 C-labeled essential amino acid, quantifying rates of protein synthesis in vivo is more challenging. The amount of isotope tracer that can be administered into the body is relatively small compared to the existing protein, thus requiring more sensitive detection. And the precursor/product labeling relationship is more complicated to interpret. The purpose of this review is to provide an overview of the principles of in vivo protein turnover studies using deuterium water (2 H2 O) with an emphasis on targeted protein analysis by hybrid LC-MS assay platforms. The pursuit of these opportunities will facilitate drug discovery and research in preclinical and clinical stages.
    Keywords:  Heavy water; LC-MS; Metabolic labeling; Protein turnover
  19. Front Mol Biosci. 2022 ;9 1084060
      A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.
    Keywords:  GC-MS; mammalian cells; metabolite extraction; metabolomics; stable isotope labeling
  20. Cell Rep. 2023 Jan 04. pii: S2211-1247(22)01851-4. [Epub ahead of print]42(1): 111950
      Advanced mass spectrometry methods have detected thousands of post-translational phosphorylation and acetylation sites in bacteria, but their functional role and the enzymes catalyzing these modifications remain largely unknown. In addition to enzymatic acetylation, lysine residues can also be chemically acetylated by the metabolite acetyl phosphate. In Escherichia coli, acetylation at over 3,000 sites has been linked to acetyl phosphate, but the functionality of this widespread non-enzymatic acetylation is even less clear than the enzyme-catalyzed one. Here, we investigate the role of acetyl-phosphate-mediated acetylation in E. coli central metabolism. Out of 19 enzymes investigated, only GapA and GpmA are acetylated at high stoichiometry, which inhibits their activity by interfering with substrate binding, effectively reducing glycolysis when flux to or from acetate is high. Extrapolating our results to the whole proteome, maximally 10% of the reported non-enzymatically acetylated proteins are expected to reach a stoichiometry that could inhibit their activity.
    Keywords:  CP: Microbiology; mass spectrometry; metabolism; post-translational modifications; post-translational regulation; protein acetylation; systems biology
  21. Anal Biochem. 2023 Jan 07. pii: S0003-2697(23)00001-5. [Epub ahead of print] 115036
      Flow-injection mass spectrometry (FI-MS) enables metabolomics studies with a very high sample-throughput. However, FI-MS is prone to in-source modifications of analytes because samples are directly injected into the electrospray ionization source of a mass spectrometer without prior chromatographic separation. Here, we spiked authentic standards of 160 primary metabolites individually into an Escherichia coli metabolite extract and measured the thus derived 160 spike-in samples by FI-MS. Our results demonstrate that FI-MS can capture a wide range of chemically diverse analytes within 30 seconds measurement time. However, the data also revealed extensive in-source modifications. Across all 160 spike-in samples, we identified significant increases of 11,013 ion peaks in positive and negative mode combined. To explain these unknown m/z features, we connected them to the m/z feature of the (de-)protonated metabolite using information about mass differences and MS2 spectra. This resulted in networks that explained on average 49% of all significant features. The networks showed that a single metabolite undergoes compound specific and often sequential in-source modifications like adductions, chemical reactions, and fragmentations. Our results show that FI-MS generates complex MS1 spectra, which leads to an overestimation of significant features, but neutral losses and MS2 spectra explain many of these features.
    Keywords:  Electrospray ionization; Feature network; Flow-injection mass spectrometry; In-source modifications; Metabolomics
  22. Front Cell Dev Biol. 2022 ;10 1032360
      Tumor-infiltrating immune cells experience significant metabolic reprogramming in the tumor microenvironment (TME), and they share similar metabolic pathways and nutrient needs with malignant cells. This positions these cell types in direct nutrient competition in the TME. We currently lack a complete understanding of the similarities, differences, and functional consequences of the metabolic pathways utilized by activated immune cells from different lineages versus neoplastic cells. This study applies a novel in situ approach using implantable microdevices to expose the tumor to 27 controlled and localized metabolic perturbations in order to perform a systematic investigation into the metabolic regulation of the cellular fitness and persistence between immune and tumor cells directly within the native TME. Our findings identify the most potent metabolites, notably glutamine and arginine, that induce a favorable metabolic immune response in a mammary carcinoma model, and reveal novel insights on less characterized pathways, such as cysteine and glutathione. We then examine clinical samples from cancer patients to confirm the elevation of these pathways in tumor regions that are enriched in activated T cells. Overall, this work provides the first instance of a highly multiplexed in situ competition assay between malignant and immune cells within tumors using a range of localized microdose metabolic perturbations. The approach and findings may be used to potentiate the effects of T cell stimulating immunotherapies on a tumor-specific or personalized basis through targeted enrichment or depletion of specific metabolites.
    Keywords:  T-cells infiltration; cancer metabolism; immunometabolism; immunotherapy; in situ perturbation; tumor micro-environment
  23. G3 (Bethesda). 2023 Jan 11. pii: jkad005. [Epub ahead of print]
      Enzymes of one-carbon metabolism play pivotal roles in proliferating cells. They are involved in the metabolism of amino acids, nucleotides, and lipids and the supply of all cellular methylations. However, there is limited information about how these enzymes are regulated during cell division and how cell cycle kinetics are affected in several loss-of-function mutants of one-carbon metabolism. Here, we report that the levels of the S. cerevisiae enzymes Ade17p and Cho2p, involved in the de novo synthesis of purines and phosphatidylcholine, respectively, are cell cycle-regulated. Cells lacking Ade17p, Cho2p, or Shm2p (an enzyme that supplies one-carbon units from serine) have distinct alterations in size homeostasis and cell cycle kinetics. Loss of Ade17p leads to a specific delay at START, when cells commit to a new round of cell division, while loss of Shm2p has broader effects, reducing growth rate. Furthermore, the inability to synthesize phosphatidylcholine de novo in cho2Δ cells delays START and reduces the coherence of nuclear elongation late in the cell cycle. Loss of Cho2p also leads to profound metabolite changes. Besides the expected changes in the lipidome, cho2Δ cells have reduced levels of amino acids, resembling cells shifted to poorer media. These results reveal the different ways that one-carbon metabolism allocates resources to affect cell proliferation at multiple cell cycle transitions.
    Keywords:   ADE17 ; CHO2 ; SHM2 ; cell size; nuclear division; phosphatidylcholine
  24. Endocrinology. 2023 Jan 13. pii: bqad003. [Epub ahead of print]
    Keywords:  Adrenal; Cancer; Cholesterol; Lipid