bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒03‒07
forty-eight papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Bio Protoc. 2019 Nov 20. 9(22): e3427
      Cancer is a disease characterized by altered metabolism, and there has been renewed interest in understanding the metabolism of tumors. Even though nutrient availability is a critical determinant of tumor metabolism, there has been little systematic study of the nutrients directly available to cancer cells in the tumor microenvironment. Previous work characterizing the metabolites present in the tumor interstitial fluid has been restricted to the measurement of a small number of nutrients such as glucose and lactate in a limited number of samples. Here we adapt a centrifugation-based method of tumor interstitial fluid isolation readily applicable to a number of sample types and a mass spectrometry-based method for the absolute quantitation of many metabolites in interstitial fluid samples. In this method, tumor interstitial fluid (TIF) is analyzed by liquid chromatography-mass spectrometry (LC/MS) using both isotope dilution and external standard calibration to derive absolute concentrations of targeted metabolites present in interstitial fluid. The use of isotope dilution allows for accurate absolute quantitation of metabolites, as other methods of quantitation are inadequate for determining nutrient concentrations in biological fluids due to matrix effects that alter the apparent concentration of metabolites depending on the composition of the fluid in which they are contained. This method therefore can be applied to measure the absolute concentrations of many metabolites in interstitial fluid from diverse tumor types, as well as most other biological fluids, allowing for characterization of nutrient levels in the microenvironment of solid tumors.
    Keywords:  Cancer metabolism; Interstitial fluid; Mass spectrometry; Metabolomics; Microenvironment; Nutrients
  2. Metabolites. 2021 Feb 25. pii: 135. [Epub ahead of print]11(3):
      Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.
    Keywords:  DHA; chromatography; lipidomics; mass spectrometry; metabolic engineering; metabolomics; thraustochytrids
  3. Metabolites. 2021 Feb 18. pii: 117. [Epub ahead of print]11(2):
      Itaconate is a small molecule metabolite that is endogenously produced by cis-aconitate decarboxylase-1 (ACOD1) in mammalian cells and influences numerous cellular processes. The metabolic consequences of itaconate in cells are diverse and contribute to its regulatory function. Here, we have applied isotope tracing and mass spectrometry approaches to explore how itaconate impacts various metabolic pathways in cultured cells. Itaconate is a competitive and reversible inhibitor of Complex II/succinate dehydrogenase (SDH) that alters tricarboxylic acid (TCA) cycle metabolism leading to succinate accumulation. Upon activation with coenzyme A (CoA), itaconyl-CoA inhibits adenosylcobalamin-mediated methylmalonyl-CoA (MUT) activity and, thus, indirectly impacts branched-chain amino acid (BCAA) metabolism and fatty acid diversity. Itaconate, therefore, alters the balance of CoA species in mitochondria through its impacts on TCA, amino acid, vitamin B12, and CoA metabolism. Our results highlight the diverse metabolic pathways regulated by itaconate and provide a roadmap to link these metabolites to potential downstream biological functions.
    Keywords:  TCA cycle metabolism; acetyl-CoA; branched-chain amino acids (BCAA); isotopic tracing; itaconate; itaconyl-CoA; methylmalonate; odd-chain fatty acids (OCFAs); propionyl-CoA; succinate; succinate dehydrogenase; vitamin B12
  4. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2003014118. [Epub ahead of print]118(10):
      Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.
    Keywords:  epigenetics; glutamine synthetase; mTORC1; nutrient deprivation; pancreatic cancer
  5. Metabolites. 2021 Feb 11. pii: 103. [Epub ahead of print]11(2):
      Related metabolites can be grouped into sets in many ways, e.g., by their participation in series of chemical reactions (forming metabolic pathways), or based on fragmentation spectral similarities or shared chemical substructures. Understanding how such metabolite sets change in relation to experimental factors can be incredibly useful in the interpretation and understanding of complex metabolomics data sets. However, many of the available tools that are used to perform this analysis are not entirely suitable for the analysis of untargeted metabolomics measurements. Here, we present PALS (Pathway Activity Level Scoring), a Python library, command line tool, and Web application that performs the ranking of significantly changing metabolite sets over different experimental conditions. The main algorithm in PALS is based on the pathway level analysis of gene expression (PLAGE) factorisation method and is denoted as mPLAGE (PLAGE for metabolomics). As an example of an application, PALS is used to analyse metabolites grouped as metabolic pathways and by shared tandem mass spectrometry fragmentation patterns. A comparison of mPLAGE with two other commonly used methods (overrepresentation analysis (ORA) and gene set enrichment analysis (GSEA)) is also given and reveals that mPLAGE is more robust to missing features and noisy data than the alternatives. As further examples, PALS is also applied to human African trypanosomiasis, Rhamnaceae, and American Gut Project data. In addition, normalisation can have a significant impact on pathway analysis results, and PALS offers a framework to further investigate this. PALS is freely available from our project Web site.
    Keywords:  Mass2Motif; SVD; liquid chromatography–mass spectrometry (LC/MS); matrix decomposition; metabolite sets; molecular family; pathways
  6. Metabolomics. 2021 Mar 03. 17(3): 29
      INTRODUCTION: Osteoarthritis (OA) is a common cause of disability in older people, but its aetiology is not yet fully understood. Biomarkers of OA from metabolomics studies have shown potential use in understanding the progression and pathophysiology of OA.OBJECTIVES: To investigate possible surrogate biomarkers of knee OA in urine using metabolomics to contribute towards a better understanding of OA progression and possible targeted treatment.
    METHOD: Liquid chromatography-high resolution mass spectrometry (LC-HRMS) was applied in a case-control approach to explore the possible metabolic differences between the urinary profiles of symptomatic knee OA patients (n = 74) (subclassified into inflammatory OA, n = 22 and non-inflammatory OA, n = 52) and non-OA controls (n = 68). Univariate, multivariate and pathway analyses were performed with a rigorous validation including cross-validation, permutation test, prediction and receiver operating characteristic curve to identify significantly altered metabolites and pathways in OA.
    RESULTS: OA datasets generated 7405 variables and multivariate analysis showed clear separation of inflammatory OA, but not non-inflammatory OA, from non-OA controls. Adequate cross-validation (R2Y = 0.874, Q2 = 0.465) was obtained. The prediction model and the ROC curve showed satisfactory results with a sensitivity of 88%, specificity of 71% and accuracy of 77%. 26 metabolites were identified as potential biomarkers of inflammatory OA using HMDB, authentic standards and/or MS/MS database.
    CONCLUSION: Urinary metabolic profiles were altered in inflammatory knee OA subjects compared to those with non-inflammatory OA and non-OA controls. These altered profiles associated with perturbed activity of the TCA cycle, pyruvate and amino acid metabolism linked to inflammation, oxidative stress and collagen destruction. Of note, 2-keto-glutaramic acid level was > eightfold higher in the inflammatory OA patients compared to non-OA control, signalling a possible perturbation in glutamine metabolism related to OA progression.
    Keywords:  Biomarkers; HILIC; LC-HRMS; Osteoarthritis; Untargeted metabolomics
  7. Metabolites. 2021 Feb 26. pii: 137. [Epub ahead of print]11(3):
      Oxylipins derived from omega-3 and -6 fatty acids are actively involved in inflammatory and immune processes and play important roles in human disease. However, as the interest in oxylipins increases, questions remain regarding which molecules are detectable in plasma, the best methods of collecting samples, and if molecules are stable during collection and storage. We thereby built upon existing studies by examining the stability of an expanded panel of 90 oxylipins, including specialized pro-resolving lipid mediators (SPMs), in human plasma (n = 5 subjects) during sample collection, processing, and storage at -80 °C. Oxylipins were quantified using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Blood samples collected in ethylenediaminetetraacetic acid (EDTA) or heparin followed by up to 2 h at room temperature prior to processing showed no significant differences in oxylipin concentrations compared to immediately processed samples, including the SPMs lipoxin A4 and resolvin D1. The majority of molecules, including SPMs, remained stable following storage for up to 1 year. However, in support of previous findings, changes were seen in a small subset of oxylipins including 12-HETE, TXB2, 14-HDHA, and 18-HEPE. Overall, this study showed that accurate measurements of most oxylipins can be obtained from stored EDTA or heparin plasma samples using LC/MS/MS.
    Keywords:  blood processing; lipid mediators; oxylipins; plasma; storage
  8. Cancers (Basel). 2021 Feb 26. pii: 978. [Epub ahead of print]13(5):
      MACC1 is a prognostic and predictive metastasis biomarker for more than 20 solid cancer entities. However, its role in cancer metabolism is not sufficiently explored. Here, we report on how MACC1 impacts the use of glucose, glutamine, lactate, pyruvate and fatty acids and show the comprehensive analysis of MACC1-driven metabolic networks. We analyzed concentration-dependent changes in nutrient use, nutrient depletion, metabolic tracing employing 13C-labeled substrates, and in vivo studies. We found that MACC1 permits numerous effects on cancer metabolism. Most of those effects increased nutrient uptake. Furthermore, MACC1 alters metabolic pathways by affecting metabolite production or turnover from metabolic substrates. MACC1 supports use of glucose, glutamine and pyruvate via their increased depletion or altered distribution within metabolic pathways. In summary, we demonstrate that MACC1 is an important regulator of metabolism in cancer cells.
    Keywords:  MACC1; cancer metabolism; colorectal cancer; metabolic networks; metabolic profiling
  9. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2020187118. [Epub ahead of print]118(10):
      Identification of common patterns of cancer metabolic reprogramming could assist the development of new therapeutic strategies. Recent attention in this field has focused on identifying and targeting signal transduction pathways that interface directly with major metabolic control processes. In the current study we demonstrate the importance of signaling by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to the metabolism and proliferation of the HCT116 colonic tumor cell line. We observed reciprocal cross talk between PPIP5K catalytic activity and glucose metabolism, and we show that CRISPR-mediated PPIP5K deletion suppresses HCT116 cell proliferation in glucose-limited culture conditions that mimic the tumor cell microenvironment. We conducted detailed, global metabolomic analyses of wild-type and PPIP5K knockout (KO) cells by measuring both steady-state metabolite levels and by performing isotope tracing experiments. We attribute the growth-impaired phenotype to a specific reduction in the supply of precursor material for de novo nucleotide biosynthesis from the one carbon serine/glycine pathway and the pentose phosphate pathway. We identify two enzymatic control points that are inhibited in the PPIP5K KO cells: serine hydroxymethyltransferase and phosphoribosyl pyrophosphate synthetase, a known downstream target of AMP-regulated protein kinase, which we show is noncanonically activated independently of adenine nucleotide status. Finally, we show the proliferative defect in PPIP5K KO cells can be significantly rescued either by addition of inosine monophosphate or a nucleoside mixture or by stable expression of PPIP5K activity. Overall, our data describe multiple, far-reaching metabolic consequences for metabolic supervision by PPIP5Ks in a tumor cell line.
    Keywords:  PPIP5K; inositol pyrophosphates; nucleotide synthesis; pentose phosphate pathway; serine–glycine–one-carbon metabolism
  10. Pharmacol Ther. 2021 Mar 01. pii: S0163-7258(21)00029-2. [Epub ahead of print] 107827
      Metabolic reprogramming is a hallmark of cancer and increasing evidence suggests that reprogrammed cell metabolism supports tumor initiation, progression, metastasis and drug resistance. Understanding metabolic dysregulation may provide therapeutic targets and facilitate drug research and development for cancer therapy. Metabolomics enables the high-throughput characterization of a large scale of small molecule metabolites in cells, tissues and biofluids, while metabolic flux analysis (MFA) tracks dynamic metabolic activities using stable isotope tracer methods. Recent advances in metabolomics and MFA technologies make them powerful tools for metabolic profiling and characterizing metabolic activities in health and disease, especially in cancer research. In this review, we introduce recent advances in metabolomics and MFA analytical technologies, and provide the first comprehensive summary of the most commonly used isotope tracing methods. In addition, we highlight how metabolomics and MFA are applied in cancer pharmacology studies particularly for discovering targetable metabolic vulnerabilities, understanding the mechanisms of drug action and drug resistance, exploring potential strategies with dietary intervention, identifying cancer biomarkers, as well as enabling precision treatment with pharmacometabolomics.
    Keywords:  cancer; drug discovery; metabolic flux; metabolomics; pharmacology
  11. Bio Protoc. 2020 Jul 20. 10(14): e3693
      Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) methods for optimized coverage and quantification. This allows for a comprehensive characterization of cellular metabolism that can be used to understand the impact of a variety of environmental stimuli on macrophage metabolism and function.
    Keywords:  LC-MS; Lipidomics; Macrophage; Metabolism; Metabolomics
  12. Int J Environ Res Public Health. 2021 Feb 23. pii: 2167. [Epub ahead of print]18(4):
      Proper preoperative ovarian cancer (OC) diagnosis remains challenging. Serum free amino acid (SFAA) profiles were investigated to identify potential novel biomarkers of OC and assess their performance in ovarian tumor differential diagnosis. Serum samples were divided based on the histopathological result: epithelial OC (n = 38), borderline ovarian tumors (n = 6), and benign ovarian tumors (BOTs) (n = 62). SFAA profiles were evaluated using aTRAQ methodology based on high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Levels of eleven amino acids significantly differed between OC+borderline and BOTs. The highest area under the receiver operating characteristic curve (AUC of ROC) (0.787) was obtained for histidine. Cystine and histidine were identified as best single markers for early stage OC/BOT and type I OC. For advanced stage OC, seven amino acids differed significantly between the groups and citrulline obtained the best AUC of 0.807. Between type II OC and BOTs, eight amino acids differed significantly and the highest AUC of 0.798 was achieved by histidine and citrulline (AUC of 0.778). Histidine was identified as a potential new biomarker in differential diagnosis of ovarian tumors. Adding histidine to a multimarker panel together with CA125 and HE4 improved the differential diagnosis between OC and BOTs.
    Keywords:  amino acids; biomarker; metabolic profiling; metabolomics; ovarian cancer; ovarian neoplasm; ovarian tumour
  13. Antioxidants (Basel). 2021 Feb 20. pii: 317. [Epub ahead of print]10(2):
      Inducers of ferroptosis such as the glutathione depleting agent Erastin and the GPX4 inhibitor Rsl-3 are being actively explored as potential therapeutics in various cancers, but the factors that determine their sensitivity are poorly understood. Here, we show that expression levels of both subunits of the cystine/glutamate antiporter xCT determine the expression of GPX4 in breast cancer, and that upregulation of the xCT/selenocysteine biosynthesis/GPX4 production axis paradoxically renders the cancer cells more sensitive to certain types of ferroptotic stimuli. We find that GPX4 is strongly upregulated in a subset of breast cancer tissues compared to matched normal samples, and that this is tightly correlated with the increased expression of the xCT subunits SLC7A11 and SLC3A2. Erastin depletes levels of the antioxidant selenoproteins GPX4 and GPX1 in breast cancer cells by inhibiting xCT-dependent extracellular reduction which is required for selenium uptake and selenocysteine biosynthesis. Unexpectedly, while breast cancer cells are resistant compared to nontransformed cells against oxidative stress inducing drugs, at the same time they are hypersensitive to lipid peroxidation and ferroptosis induced by Erastin or Rsl-3, indicating that they are 'addicted' to the xCT/GPX4 axis. Our findings provide a strategic basis for targeting the anti-ferroptotic machinery of breast cancer cells depending on their xCT status, which can be further explored.
    Keywords:  Erastin; GPX4; Rsl-3; breast cancer; ferroptosis; lipid peroxidation; selenium
  14. J Lipid Res. 2021 Feb 26. pii: S0022-2275(21)00038-9. [Epub ahead of print] 100056
      Methionine is an essential amino acid and critical precursor to the cellular methyl donor S-adenosylmethionine. Unlike non-transformed cells, cancer cells have a unique metabolic requirement for methionine and are unable to proliferate in growth media where methionine is replaced with its metabolic precursor, homocysteine. This metabolic vulnerability is common among cancer cells regardless of tissue origin and is known as "methionine dependence", "methionine stress sensitivity", or the Hoffman effect. The response of lipids to methionine stress, however, is not well-understood. Using mass spectroscopy, label-free vibrational microscopy, and next-generation sequencing, we characterize the response of lipids to methionine stress in the triple-negative breast cancer cell line MDA-MB-468 and its methionine stress insensitive derivative, MDA-MB-468res-R8. Lipidome analysis identified an immediate, global decrease in lipid abundances with the exception of triglycerides and an increase in lipid droplets in response to methionine stress specifically in MDA-MB-468 cells. Furthermore, specific gene expression changes were observed as a secondary response to methionine stress in MDA-MB-468, resulting in a downregulation of fatty acid metabolic genes and an upregulation of genes in the unfolded protein response pathway. We conclude that the extensive changes in lipid abundance during methionine stress is a direct consequence of the modified metabolic profile previously described in methionine stress sensitive cells. The changes in lipid abundance likely results in changes in membrane composition inducing the unfolded protein response we observe.
    Keywords:  Cancer metabolism; Fatty acid metabolism; Homocysteine; Lipid droplets; Lipid metabolism; Methionine; Methionine stress; Phospholipids; Triglycerides
  15. Anal Chem. 2021 Mar 03.
      In vitro noncontact cell-based coculture models are frequently employed to study cell-to-cell communication. However, these models cannot accurately represent the complexity of in vivo signaling. d-Lactate is an unusual metabolite produced and released by cancer cells. The characterization of d-lactate is challenging as it shares the same mass but has much lower amounts compared with l-lactate. Herein, d-α-hydroxy acids were specifically recognized and dehydrogenated by d-α-hydroxy acid dehydrogenase. The dehydrogenation products were rapidly quaternized for enhancement of mass signals. An on-probe enzymatic dehydrogenation-derivatization method was proposed for chiral analysis of α-hydroxy acids at the single-cell level. It is a promising amplification methodology and affords over 3 orders of magnitude signal enhancement. Furthermore, direct contact coculture models were used to precisely mimic the tumor microenvironment and explore the communication between cancer and normal cells. Single-cell mass spectrometry (SCMS) was further applied to easily sample cell extracts and study the differences of the aspects of small molecule metabolism in cocultured cells. On the basis of direct contact coculture SCMS, several differential small molecule metabolites and differences of oxidative stress between cocultured and monocultured normal cells were successfully detected. Additionally, d-lactate was discovered as a valuable differential metabolite with application of the two developed methods. It may account for the cancer-associated metabolic behavior of normal cells. These changes could be relieved after d-lactate metabolism-related drug treatment. This discovery may promote the investigation of d-lactate metabolism, which may provide a novel direction for cancer therapy.
  16. Metabolites. 2021 Feb 19. pii: 119. [Epub ahead of print]11(2):
      Sporadic colorectal cancer is characterized by a multistep progression from normal epithelium to precancerous low-risk and high-risk adenomas to invasive cancer. Yet, the underlying molecular mechanisms of colorectal carcinogenesis are not completely understood. Within the "Metabolomic profiles throughout the continuum of colorectal cancer" (MetaboCCC) consortium we analyzed data generated by untargeted, mass spectrometry-based metabolomics using plasma from 88 colorectal cancer patients, 200 patients with high-risk adenomas and 200 patients with low-risk adenomas recruited within the "Colorectal Cancer Study of Austria" (CORSA). Univariate logistic regression models comparing colorectal cancer to adenomas resulted in 442 statistically significant molecular features. Metabolites discriminating colorectal cancer patients from those with adenomas in our dataset included acylcarnitines, caffeine, amino acids, glycerophospholipids, fatty acids, bilirubin, bile acids and bacterial metabolites of tryptophan. The data obtained discovers metabolite profiles reflecting metabolic differences between colorectal cancer and colorectal adenomas and delineates a potentially underlying biological interpretation.
    Keywords:  adenoma; colorectal cancer; metabolite profiling; untargeted metabolomics
  17. Elife. 2021 Mar 01. pii: e64806. [Epub ahead of print]10
      Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologues in snake venoms (LAAO, L-amino acid oxidases), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell productive gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.
    Keywords:  cell biology; human; immunology; inflammation; mouse
  18. Biomolecules. 2021 Feb 21. pii: 326. [Epub ahead of print]11(2):
      Obesity is an increasing public health concern both in the developed and developing countries. Previous studies have demonstrated that considerable alterations in lipid metabolism and consequently marked changes in lipid profile are associated with the onset and progression of obesity-related complications. To characterize the full spectrum of obesity-induced changes in lipid metabolism, direct infusion tandem mass spectrometry analysis is the most promising approach. To better understand which of the many lipid species are the most strongly associated with obesity, the aim of our work was to measure and profile plasma lipids in normal (n = 57), overweight (n = 31), and obese (n = 48) individuals randomly selected from samples of Hungarian general and Roma populations by using the targeted quantitative lipidomics platform, the Lipidyzer. Principal component and stepwise regression analyses were used to identify the most significant clusters and species of lipids by increasing body mass index (BMI). From the 18 clusters identified four key lipid species (PE P-16:0/20:3, TG 20:4_33:1, TG 22:6_36:4, TG 18:3_33:0) showed a strong significant positive and three others (Hex-Cer 18:1;O2/22:0, LPC 18:2, PC 18:1_18:1) significant negative association with BMI. Compared to individual lipid species alone, the lipid species ratio (LSR) we introduced showed an extremely strong, at least 9 orders of magnitude stronger, association with BMI. The LSR can be used as a sensitive and predictive indicator to monitor obesity-related alterations in human plasma and control the effectiveness of treatment of obesity associated non-communicable diseases.
    Keywords:  Lipidyzer platform; body mass index (BMI), lipidomic analysis; exploratory principal component analysis; lipid species ratio; obesity; stepwise regression analysis
  19. Acta Pharmacol Sin. 2021 Mar 03.
      Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 μg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.
    Keywords:  NAC; P-glycoprotein; Rosup; cystine; docetaxel; drug resistance; metabolomics; microenvironment; non-small cell lung cancer cells; redox homeostasis
  20. Amino Acids. 2021 Feb 28.
      Dysregulated cellular energetics has recently been recognized as a hallmark of cancer and garnered attention as a potential targeting strategy for cancer therapeutics. Cancer cells reprogram metabolic activities to meet bio-energetic, biosynthetic and redox requirements needed to sustain indefinite proliferation. In many cases, metabolic reprogramming is the result of complex interactions between genetic alterations in well-known oncogenes and tumor suppressors and epigenetic changes. While the metabolism of the two most abundant nutrients, glucose and glutamine, is reprogrammed in a wide range of cancers, accumulating evidence demonstrates that additional metabolic pathways are also critical for cell survival and growth. Proline metabolism is one such metabolic pathway that promotes tumorigenesis in multiple cancer types, including liver cancer, which is the fourth main cause of cancer mortality in the world. Despite the recent spate of approved treatments, including targeted therapy and combined immunotherapies, there has been no significant gain in clinical benefits in the majority of liver cancer patients. Thus, exploring novel therapeutic strategies and identifying new molecular targets remains a top priority for liver cancer. Two of the enzymes in the proline biosynthetic pathway, pyrroline-5-carboxylate reductase (PYCR1) and Aldehyde Dehydrogenase 18 Family Member A1 (ALDH18A1), are upregulated in liver cancer of both human and animal models, while proline catabolic enzymes, such as proline dehydrogenase (PRODH) are downregulated. Here we review the latest evidence linking proline metabolism to liver and other cancers and potential mechanisms of action for the proline pathway in cancer development.
    Keywords:  Cancer; Enzyme; Hepatocellular carcinoma; Metabolism; Proline
  21. Arch Toxicol. 2021 Mar 02.
      A fundamental metabolic feature of cancerous tissues is high glucose consumption. The rate of glucose consumption in a cancer cell can be 10-15 times higher than in normal cells. Isolation and cultivation of tumor cells in vitro highlight properties that are associated with intensive glucose utilization, the presence of minimal oxidative metabolism, an increase in lactate concentrations in the culture medium and a reduced rate of oxygen consumption. Although glycolysis is suggested as a general feature of malignant cells and recently identified as a possible contributing factor to tumor progression, several studies highlight distinct metabolic characteristics in some tumors, including a relative decrease in avidity compared to glucose and/or a glutamine dependency of lactate and even proliferative tumor cells. The aim of this review is to determine the particularities in the energy metabolism of cancer cells, focusing on the main nutritional substrates, such as glucose and glutamine, evaluating lactate dehydrogenase as a potential marker of malignancy and estimating activators and inhibitors in cancer treatment.
    Keywords:  Activators; Cancer; Glucose; Glycolysis; Inhibitors; Krebs cycle
  22. Anal Chem. 2021 Mar 02.
      Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three μg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (μef) of unmodified histone proteoforms can be predicted accurately (R2 = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.
  23. Bio Protoc. 2020 Sep 20. 10(18): e3756
      Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.
    Keywords:  Advantage over Liquid-Chromatography (LC); Chromatin; Direct injection; Histone; Mass spectrometry; Post-translational modifications (PTMs)
  24. Anal Chem. 2021 Mar 01.
      Single-cell capillary electrophoresis mass spectrometry (CE-MS) is a promising platform to analyze cellular contents and probe cell heterogeneity. However, current single-cell CE-MS methods often rely on offline microsampling processes and may demonstrate low sampling precision and accuracy. We have recently developed an electrospray-assisted device, spray-capillary, for low-volume sample extraction. With the spray-capillary, low-volume samples (pL-nL) are drawn into the sampling end of the device, which can be used directly for CE separation and online MS detection. Here, we redesigned the spray-capillary by utilizing a capillary with a <15 μm tapered tip so that it can be directly inserted into single cells for sample collection and on-capillary CE-MS analysis. We evaluated the performance of the modified spray-capillary by performing single-cell microsampling on single onion cells with varying sample injection times and direct MS analysis or online CE-MS analysis. We have demonstrated, for the first time, online sample collection and CE-MS for the analysis of single cells. This application of the modified spray-capillary device facilitates the characterization and relative quantification of hundreds of metabolites in single cells.
  25. Bio Protoc. 2020 May 05. 10(9): e3613
      In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in biological samples, gas chromatography-hydrogen flame ionization detection (GC-FID)-based methods have been used as highly robust and reliable techniques. Prior to GC-FID analysis, FAs need to be derivatized to volatile FA methyl esters (FAMEs). The derivatization of unsaturated FAs using classical derivatization methods that rely on high reaction temperature requires skill; consequently, the quantification results are often unreliable. The recently available FA-methylation procedure rapidly and reliably derivatizes a variety of FA species, including poly-unsaturated FAs (PUFAs). To analyze FAs in mammalian tissue samples, lipid extraction and fractionation are also critical for robust analysis. In this report, we describe a whole protocol for the GC-FID-based FA quantification of mammalian tissue samples, including lipid extraction, fractionation, derivatization, and quantification. The protocol is useful when various FAs, especially unsaturated FAs, need to be reliably quantified.
    Keywords:  Fatty acid methyl esters; Fatty acids; Flame ionization detection; Gas chromatography; Lipidomics; Quantification
  26. Bioanalysis. 2021 Mar 04.
      Background: The acetate ion has important physiological functions and important therapeutic applications. A rapid LC-MS/MS method is described to measure acetate ions in human plasma without chemical derivatization. Materials & methods: A 200 μl sample was spiked with the internal standard 1,2-13C-acetate and proteins precipitated with trichloroacetic acid. The supernatant was recovered and separated under acidic conditions on a C18-column. The eluent was alkalinized by post-column infusion of methanolic ammonium hydroxide. Acetate ions were monitored on a low resolution mass spectrometer in negative ion mode. Results: Method was validated for accuracy and precision with a lower limit of quantitation of 9.7 μM and linear dynamic range up to 339.6 μM. Conclusion: The method is open for analytical improvement and adapts with metabolomic and pharmacometabolomic studies on chemicals of similar nature.
    Keywords:  acetate; acetic acid toxicity; mass spectrometry; metabolomics; pharmacometabolomics; pseudo multiple reaction monitoring; short chain fatty acids
  27. Metabolites. 2021 Feb 26. pii: 140. [Epub ahead of print]11(3):
      Glycosphingolipids (GSL) represent a highly heterogeneous class of lipids with many cellular functions, implicated in a wide spectrum of human diseases. Their isolation, detection, and comprehensive structural analysis is a challenging task due to the structural diversity of GSL molecules. In this work, GSL subclasses are isolated from human plasma using an optimized monophasic ethanol-water solvent system capable to recover a broad range of GSL species. Obtained deproteinized plasma is subsequently purified and concentrated by C18-based solid-phase extraction (SPE). The hydrophilic interaction liquid chromatography coupled to electrospray ionization linear ion trap tandem mass spectrometry (HILIC-ESI-LIT-MS/MS) is used for GSL analysis in the human plasma extract. Our results provide an in-depth profiling and structural characterization of glycosphingolipid and some phospholipid subclasses identified in the human plasma based on their retention times and the interpretation of tandem mass spectra. The structural composition of particular lipid species is readily characterized based on the detailed interpretation of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) spectra and further confirmed by specific fragmentation behavior following predictable patterns, which yields to the unambiguous identification of 154 GSL species within 7 lipid subclasses and 77 phospholipids representing the highest number of GSL species ever reported in the human plasma. The developed HILIC-ESI-MS/MS method can be used for further clinical and biological research of GSL in the human blood or other biological samples.
    Keywords:  fragmentation behavior; glycosphingolipids; human plasma; hydrophilic interaction liquid chromatography; lipid profile; lipidomics; mass spectrometry; sample preparation
  28. Bio Protoc. 2020 Jul 05. 10(13): e3670
      Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.
    Keywords:  Cholesterol; Lipid rafts; Lipidomics; Lipids; Membrane
  29. Curr Protoc. 2021 Mar;1(3): e69
      Data-independent-acquisition mass spectrometry (DIA-MS) is a state-of-the-art proteomic technique for high-throughput identification and quantification of peptides and proteins. Interpretation of DIA-MS data relies on the use of a spectral library, which is optimally created from data acquired from the same samples in data-dependent acquisition (DDA) mode. As DIA-MS quantification relies on the spectral libraries, having a high-quality, non-redundant, and comprehensive spectral library is essential. This article describes the major steps for creating a high-quality spectral library using a combination of multiple complementary search engines. We discuss appropriate strategies to control the false discovery rate for the final spectral library as a result of merging multiple searches. © 2021 The Authors Current Protocols © 2021 Wiley Periodicals LLC. Basic Protocol 1: Searching DDA-MS files with multiple search engines Basic Protocol 2: Merging results from multiple search engines Basic Protocol 3: Creating spectral libraries from merged results Alternate Protocol: Using CLI for automating tasks Support Protocol: Creating concatenated FASTA files.
    Keywords:  DIA; SWATH; mass spectrometry; proteomics; spectral library
  30. Cancer Discov. 2021 Mar 02. pii: candisc.0119.2020. [Epub ahead of print]
      Although pancreatic ductal adenocarcinoma (PDAC) cells are exposed to a nutrient-depleted tumor microenvironment, they can acquire nutrients via macropinocytosis, an endocytic form of protein scavenging that functions to support cancer metabolism. Here, we provide evidence that macropinocytosis is operational in the pancreatic tumor stroma. We find that glutamine deficiency triggers macropinocytic uptake in pancreatic cancer-associated fibroblasts (CAFs). Mechanistically, we decipher that stromal macropinocytosis is potentiated via the enhancement of cytosolic Ca2+ and dependent on ARHGEF2 and CaMKK2-AMPK signaling. We elucidate that macropinocytosis has dual function in CAFs - it serves as a source of intracellular amino acids that sustain CAF cell fitness and function, and it provides secreted amino acids that promote tumor cell survival. Importantly, we demonstrate that stromal macropinocytosis supports PDAC tumor growth. These results highlight the functional role of macropinocytosis in the tumor stroma and provide a mechanistic understanding of how nutrient deficiency can control stromal protein scavenging.
  31. Cancers (Basel). 2021 Feb 12. pii: 762. [Epub ahead of print]13(4):
      The specific molecular features of cancer cells that distinguish them from the normal ones are denoted as "hallmarks of cancer". One of the critical hallmarks of cancer is an altered metabolism which provides tumor cells with energy and structural resources necessary for rapid proliferation. The key feature of a cancer-reprogrammed metabolism is its plasticity, allowing cancer cells to better adapt to various conditions and to oppose different therapies. Furthermore, the alterations of metabolic pathways in malignant cells are heterogeneous and are defined by several factors including the tissue of origin, driving mutations, and microenvironment. In the present review, we discuss the key features of metabolic reprogramming and plasticity associated with different stages of tumor, from primary tumors to metastases. We also provide evidence of the successful usage of metabolic drugs in anticancer therapy. Finally, we highlight new promising targets for the development of new metabolic drugs.
    Keywords:  aerobic glycolysis; cancer metabolism; cancer therapy; lipid metabolism; metabolic reprograming; one-carbon metabolism
  32. J Proteome Res. 2021 Mar 03.
      Every laboratory performing mass-spectrometry-based proteomics strives to generate high-quality data. Among the many factors that impact the outcome of any experiment in proteomics is the LC-MS system performance, which should be monitored within each specific experiment and also long term. This process is termed quality control (QC). We present an easy-to-use tool that rapidly produces a visual, HTML-based report that includes the key parameters needed to monitor the LC-MS system performance, with a focus on monitoring the performance within an experiment. The tool, named RawBeans, generates a report for individual files or for a set of samples from a whole experiment. We anticipate that it will help proteomics users and experts evaluate raw data quality independent of data processing. The tool is available at The mass-spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD022816.
    Keywords:  QC; Raw Data; nanoLC-MS/MS; quality control
  33. J Am Soc Mass Spectrom. 2021 Mar 03.
      Biological systems are composed of heterogeneous populations of cells that intercommunicate to form a functional living tissue. Biological function varies greatly across populations of cells, as each single cell has a unique transcriptome, proteome, and metabolome that translates to functional differences within single species and across kingdoms. Over the past decade, substantial advancements in our ability to characterize omic profiles on a single cell level have occurred, including in multiple spectroscopic and mass spectrometry (MS)-based techniques. Of these technologies, spatially resolved mass spectrometry approaches, including mass spectrometry imaging (MSI), have shown the most progress for single cell proteomics and metabolomics. For example, reporter-based methods using heavy metal tags have allowed for targeted MS investigation of the proteome at the subcellular level, and development of technologies such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) now mean that dynamic metabolomics can be performed in situ. In this Perspective, we showcase advancements in single cell spatial metabolomics and proteomics over the past decade and highlight important aspects related to high-throughput screening, data analysis, and more which are vital to the success of achieving proteomic and metabolomic profiling at the single cell scale. Finally, using this broad literature summary, we provide a perspective on how the next decade may unfold in the area of single cell MS-based proteomics and metabolomics.
    Keywords:  DESI; LAESI; LDI; MALDI; NanoDESI; SIMS; high-throughput omics; mass spectrometry imaging
  34. Anal Chem. 2021 Mar 02.
      Spatially resolved ambient mass spectrometry imaging methods have gained popularity to characterize cancer sites and their borders using molecular changes in the lipidome. This utility, however, is predicated on metabolic homogeneity at the border, which would create a sharp molecular transition at the morphometric borders. We subjected murine models of human medulloblastoma brain cancer to mass spectrometry imaging, a technique that provides a direct readout of tissue molecular content in a spatially resolved manner. We discovered a distance-dependent gradient of cancer-like lipid molecule profiles in the brain tissue within 1.2 mm of the cancer border, suggesting that a cancer-like state progresses beyond the histologic border, into the healthy tissue. The results were further corroborated using orthogonal liquid chromatography and mass spectrometry (LC-MS) analysis of selected tissue regions subjected to laser capture microdissection. LC-MS/MS analysis for robust identification of the affected molecules implied changes in a number of different lipid classes, some of which are metabolized from the essential docosahexaenoic fatty acid (DHA) present in the interstitial fluid. Metabolic molecular borders are thus not as sharp as morphometric borders, and mass spectrometry imaging can reveal molecular nuances not observed with microscopy. Caution must be exercised in interpreting multimodal imaging results stipulated on a coincidental relationship between metabolic and morphometric borders of cancer, at least within animal models used in preclinical research.
  35. Oncogene. 2021 Mar 02.
      Peroxisome Proliferator-Activated Receptor Gamma (PPARG) is one of the three members of the PPAR family of transcription factors. Besides its roles in adipocyte differentiation and lipid metabolism, we recently demonstrated an association between PPARG and metastasis in prostate cancer. In this study a functional effect of PPARG on AKT serine/threonine kinase 3 (AKT3), which ultimately results in a more aggressive disease phenotype was identified. AKT3 has previously been shown to regulate PPARG co-activator 1 alpha (PGC1α) localisation and function through its action on chromosome maintenance region 1 (CRM1). AKT3 promotes PGC1α localisation to the nucleus through its inhibitory effects on CRM1, a known nuclear export protein. Collectively our results demonstrate how PPARG over-expression drives an increase in AKT3 levels, which in turn has the downstream effect of increasing PGC1α localisation within the nucleus, driving mitochondrial biogenesis. Furthermore, this increase in mitochondrial mass provides higher energetic output in the form of elevated ATP levels which may fuel the progression of the tumour cell through epithelial to mesenchymal transition (EMT) and ultimately metastasis.
  36. J Proteome Res. 2021 Mar 04.
      Visual examination of mass spectrometry data is necessary to assess data quality and to facilitate data exploration. Graphics provide the means to evaluate spectral properties, test alternative peptide/protein sequence matches, prepare annotated spectra for publication, and fine-tune parameters during wet lab procedures. Visual inspection of LC-MS data is constrained by proteomics visualization software designed for particular workflows or vendor-specific tools without open-source code. We built PSpecteR, an open-source and interactive R Shiny web application for visualization of LC-MS data, with support for several steps of proteomics data processing, including reading various mass spectrometry files, running open-source database search engines, labeling spectra with fragmentation patterns, testing post-translational modifications, plotting where identified fragments map to reference sequences, and visualizing algorithmic output and metadata. All figures, tables, and spectra are exportable within one easy-to-use graphical user interface. Our current software provides a flexible and modern R framework to support fast implementation of additional features. The open-source code is readily available (, and a PSpecteR Docker container ( is available for easy local installation.
    Keywords:  bottom-up; peptide database search; proteomics web application; tandem mass spectrometry; top-down
  37. Metabolites. 2021 Feb 16. pii: 112. [Epub ahead of print]11(2):
      Mitochondria are central organelles that coordinate a vast array of metabolic and biologic functions important for cellular health. Amino acids are intricately linked to the bioenergetic, biosynthetic, and homeostatic function of the mitochondrion and require specific transporters to facilitate their import, export, and exchange across the inner mitochondrial membrane. Here we review key cellular metabolic outputs of eukaryotic mitochondrial amino acid metabolism and discuss both known and unknown transporters involved. Furthermore, we discuss how utilization of compartmentalized amino acid metabolism functions in disease and physiological contexts. We examine how improved methods to study mitochondrial metabolism, define organelle metabolite composition, and visualize cellular gradients allow for a more comprehensive understanding of how transporters facilitate compartmentalized metabolism.
    Keywords:  amino acids; compartmentalization; cytosol; metabolomics; mitochondria; solute carriers; transporters
  38. Elife. 2021 Mar 01. pii: e63326. [Epub ahead of print]10
      The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast (MEF) and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.
    Keywords:  cancer biology; cell biology; human; mouse; rat
  39. Nat Commun. 2021 Mar 05. 12(1): 1455
      T-cell exhaustion denotes a hypofunctional state of T lymphocytes commonly found in cancer, but how tumor cells drive T-cell exhaustion remains elusive. Here, we find T-cell exhaustion linked to overall survival in 675 hepatocellular carcinoma (HCC) patients with diverse ethnicities and etiologies. Integrative omics analyses uncover oncogenic reprograming of HCC methionine recycling with elevated 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM) to be tightly linked to T-cell exhaustion. SAM and MTA induce T-cell dysfunction in vitro. Moreover, CRISPR-Cas9-mediated deletion of MAT2A, a key SAM producing enzyme, results in an inhibition of T-cell dysfunction and HCC growth in mice. Thus, reprogramming of tumor methionine metabolism may be a viable therapeutic strategy to improve HCC immunity.
  40. Bio Protoc. 2020 Oct 20. 10(20): e3797
      Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various types of cancers. The untargeted analysis contributes to finding new biomarkers. i.e., the analysis of all detectable signals including both known and unknown metabolites extends the coverage of metabolite to be observed. However, the observed data includes thousands of peaks. Besides, non-linear migration time fluctuation and skewed peaks are caused by the sample condition. The presented pretreatment protocols of saliva samples enhance the reproducibility of migration time drift, which facilitates the matching peaks across the samples and also results in reproducible absolute concentrations of the detected metabolites. The described protocols are utilized not only for saliva but for any liquid samples with slight modifications.
    Keywords:  Cancer; Capillary electrophoresis-mass spectrometry; Metabolomics; Polyamine; Saliva
  41. Cell Metab. 2021 Feb 23. pii: S1550-4131(21)00061-9. [Epub ahead of print]
      Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly reflect metabolite availability in human blood. Here, we performed CRISPR-based screens in traditional versus human plasma-like medium (HPLM). Sets of conditionally essential genes in human cancer cell lines span several cellular processes and vary with both natural cell-intrinsic diversity and the combination of basal and serum components that comprise typical media. Notably, we traced the causes for each of three conditional CRISPR phenotypes to the availability of metabolites uniquely defined in HPLM versus conventional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.
    Keywords:  CRISPR; HPLM; conditional gene essentiality; gene-nutrient interaction; genetic screen; physiologic medium
  42. Proteomics. 2021 Mar 04. e2000094
      Extracellular vesicles (EVs) are nanometer-size lipid vesicles released by cells, which play essential biological functions in intracellular communication. Increasing evidence indicates that EVs participate in cancer development, including invasion, migration, metastasis, and cancer immune modulation. One of the key mechanisms is that EVs affect different cells in the tumor microenvironment through surface-anchor proteins and protein cargos. Moreover, proteins specifically expressed in tumor-derived EVs can be applied in cancer diagnosis and monitoring. Besides, the EV proteome also helps to understand drug resistance in cancers and to guide clinical medication. With the development of mass spectrometry and array-based multi-protein detection, the research of EV proteomics has entered a new era. The high-throughput parallel proteomic profiling based on these new platforms allows us to study the impact of EV proteome on cancer progression more comprehensively and to describe the proteomic landscape in cancers with more details. In this review, we introduce the role and function of different types of EVs in cancer progression. More importantly, we summarize the proteomic profiling of EVs based on different methods and the application of EV proteome in cancer detection and monitoring. This article is protected by copyright. All rights reserved.
    Keywords:  cancer; extracellular vesicles; mass spectrometry; proteomics
  43. Bio Protoc. 2019 May 05. 9(9): e3224
      In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation exchange chromatography method has limited sensitivity and long run times while reverse phase chromatography requires strong ion-pairing. In this method, the sample containing crosslinks is applied on a diamond hydride column using water and acetonitrile solvents containing 0.1% (w/v) formic acid. Eight crosslinks are eluted separately from the column and detected by mass spectrometry in the sub-pmol range. By using this method, it is possible to separate all crosslinks of collagen in several biological samples without the need for ion-pairing agent or derivatization for detection.
    Keywords:  Bone; Collagen; Crosslink analysis; Mass spectrometry; Silica hydride column; Skin; Urine
  44. Mol Cell. 2021 Feb 23. pii: S1097-2765(21)00095-2. [Epub ahead of print]
      O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS.
    Keywords:  O-GlcNAcylation; SRPK2; lipid synthesis; nuclear import
  45. J Proteome Res. 2021 Mar 04.
      Error estimation for differential protein quantification by label-free shotgun proteomics is challenging due to the multitude of error sources, each contributing uncertainty to the final results. We have previously designed a Bayesian model, Triqler, to combine such error terms into one combined quantification error. Here we present an interface for Triqler that takes MaxQuant results as input, allowing quick reanalysis of already processed data. We demonstrate that Triqler outperforms the original processing for a large set of both engineered and clinical/biological relevant data sets. Triqler and its interface to MaxQuant are available as a Python module under an Apache 2.0 license from
    Keywords:  Bayesian statistics; label-free quantification; mass spectrometry; proteomics; quantification
  46. Expert Rev Proteomics. 2021 Mar 02. 1-6
      ABSTACTIntroduction:Metabolomics, one of the most high-promising technologies, is the most recently developed post-genomics discipline for developing new diagnostic tests for future implementation in medicine. More than 2,000 scientific papers, using mass spectrometry-based (MS-based) metabolomics analysis for human disease diagnostics, have been published during the past two decades, and almost every metabolomics study shows high diagnostic accuracy. However, despite the great results and promising perspectives, there are currently no diagnostic tests based on metabolomics that have been approved and introduced into clinics.Areas covered:In this report, the advantages and challenges of MS-based metabolomics are discussed with a focus on its developing role in diagnostics, and the current trends in implementing metabolomics diagnostics in the clinic.Expert opinion:In the development of new clinical diagnostics tests, MS-based metabolomics has potential as both a preliminary discovery base for routine testing and a multi-test prototype, which is hoped to be introduced into clinical practice in the near future. A laboratory-developed test (LDT) is one possible way that multi-testing could be developed.
    Keywords:  Diagnostics; disease; mass spectrometry; metabolites; metabolomics
  47. Pharmaceuticals (Basel). 2021 Feb 26. pii: 190. [Epub ahead of print]14(3):
      Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Studies in hematological malignancies have shown alterations in fatty acid, folate, and amino acid metabolism pathways in cancer cells. One-carbon (1-C) metabolism is essential for numerous cancer cell functions, including protein and nucleic acid synthesis and maintaining cellular redox balance, and inhibition of the 1-C pathway has yielded several highly active drugs, such as methotrexate and 5-FU. Glutamine depletion has also emerged as a therapeutic approach for cancers that have demonstrated dependence on glutamine for survival. Recent studies have shown that in response to glutamine deprivation leukemia cells upregulate key enzymes in the serine biosynthesis pathway, suggesting that serine upregulation may be a targetable compensatory mechanism. These new findings may provide opportunities for novel cancer treatments.
    Keywords:  amino acid metabolism; amino acid restriction in cancer; amino-acid-degrading enzymes; cancer therapy; leukemia
  48. J Clin Med. 2021 Feb 16. pii: 792. [Epub ahead of print]10(4):
      Non-alcoholic fatty liver disease is one of the most common chronic liver diseases, ranging from simple steatosis to steatohepatitis, fibrosis, and cirrhosis. Its prevalence is rapidly increasing and presently affects around 25% of the general population of Western countries, due to the obesity epidemic. Liver fat accumulation induces the synthesis of specific lipid species and particularly ceramides, a sphingolipid. In turn, ceramides have deleterious effects on hepatic metabolism, a phenomenon called lipotoxicity. We review here the evidence showing the role of ceramides in non-alcoholic fatty liver disease and the mechanisms underlying their effects.
    Keywords:  NAFLD; NASH; ceramide; liver; sphingolipids; steatosis