bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒04‒26
forty-two papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research


  1. Metab Eng. 2020 Apr 21. pii: S1096-7176(20)30065-3. [Epub ahead of print]
    Lesner NP, Gokhale A, Kota K, DeBerardinis RJ, Mishra P.
      Pathogenic mutations in the mitochondrial genome (mtDNA) impair organellar ATP production, requiring mutant cells to activate metabolic adaptations for survival. Understanding how metabolism adapts to clinically relevant mtDNA mutations may provide insight into cellular strategies for metabolic flexibility. In this study, we use 13C isotope tracing and metabolic flux analysis to investigate central carbon and amino acid metabolic reprogramming in isogenic cells containing mtDNA mutations. We identify alterations in glutamine and cystine transport which indirectly regulate mitochondrial metabolism and electron transport chain function. Metabolism of cystine can promote glucose oxidation through the transsulfuration pathway and the production of α-ketobutyrate. Intriguingly, activating or inhibiting α-ketobutyrate production is sufficient to modulate both glucose oxidation and mitochondrial respiration in mtDNA mutant cells. Thus, cystine-stimulated transsulfuration serves as an adaptive mechanism linking glucose oxidation and amino acid metabolism in the setting of mtDNA mutations.
    Keywords:  Metabolic reprogramming; Transsulfuration; mtDNA mutations
    DOI:  https://doi.org/10.1016/j.ymben.2020.03.010
  2. Cancers (Basel). 2020 Apr 18. pii: E1002. [Epub ahead of print]12(4):
    Martín-Blázquez A, Jiménez-Luna C, Díaz C, Martínez-Galán J, Prados J, Vicente F, Melguizo C, Genilloud O, Pérez Del Palacio J, Caba O.
      Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers, with a 5-year survival rate of less than 5%. In fact, complete surgical resection remains the only curative treatment. However, fewer than 20% of patients are candidates for surgery at the time of presentation. Hence, there is a critical need to identify diagnostic biomarkers with potential clinical utility in this pathology. In this context, metabolomics could be a powerful tool to search for new robust biomarkers. Comparative metabolomic profiling was performed in serum samples from 59 unresectable PDAC patients and 60 healthy controls. Samples were analyzed by using an untargeted metabolomics workflow based on liquid chromatography, coupled to high-resolution mass spectrometry in positive and negative electrospray ionization modes. Univariate and multivariate analysis allowed the identification of potential candidates that were significantly altered in PDAC patients. A panel of nine candidates yielded excellent diagnostic capacities. Pathway analysis revealed four altered pathways in our patients. This study shows the potential of liquid chromatography coupled to high-resolution mass spectrometry as a diagnostic tool for PDAC. Furthermore, it identified novel robust biomarkers with excellent diagnostic capacities.
    Keywords:  biomarker; diagnosis; metabolomics; pancreatic ductal adenocarcinoma; reverse-phase liquid chromatography
    DOI:  https://doi.org/10.3390/cancers12041002
  3. Int J Mol Sci. 2020 Apr 20. pii: E2872. [Epub ahead of print]21(8):
    Ortiz-Pedraza Y, Muñoz-Bello JO, Olmedo-Nieva L, Contreras-Paredes A, Martínez-Ramírez I, Langley E, Lizano M.
      Cancer cells exhibit exacerbated metabolic activity to maintain their accelerated proliferation and microenvironmental adaptation in order to survive under nutrient-deficient conditions. Tumors display an increase in glycolysis, glutaminolysis and fatty acid biosynthesis, which provide their energy source. Glutamine is critical for fundamental cellular processes, where intermediate metabolites produced through glutaminolysis are necessary for the maintenance of mitochondrial metabolism. These include antioxidants to remove reactive oxygen species, and the generation of the nonessential amino acids, purines, pyrimidines and fatty acids required for cellular replication and the activation of cell signaling. Some cancer cells are highly dependent on glutamine consumption since its catabolism provides an anaplerotic pathway to feed the Krebs cycle. Intermediate members of the glutaminolysis pathway have been found to be deregulated in several types of cancers and have been proposed as therapeutic targets and prognostic biomarkers. This review summarizes the main players in the glutaminolysis pathway, how they have been found to be deregulated in cancer and their implications for cancer maintenance. Furthermore, non-coding RNAs are now recognized as new participants in the regulation of glutaminolysis; therefore, their involvement in glutamine metabolism in cancer is discussed in detail.
    Keywords:  cancer; glutaminolysis; lncRNAs; miRNAs
    DOI:  https://doi.org/10.3390/ijms21082872
  4. Mol Genet Metab. 2020 Apr 03. pii: S1096-7192(20)30081-0. [Epub ahead of print]
    Johnson SC, Kayser EB, Bornstein R, Stokes J, Bitto A, Park KY, Pan A, Sun G, Raftery D, Kaeberlein M, Sedensky MM, Morgan PG.
      Leigh Syndrome (LS) is a mitochondrial disorder defined by progressive focal neurodegenerative lesions in specific regions of the brain. Defects in NDUFS4, a subunit of complex I of the mitochondrial electron transport chain, cause LS in humans; the Ndufs4 knockout mouse (Ndufs4(KO)) closely resembles the human disease. Here, we probed brain region-specific molecular signatures in pre-symptomatic Ndufs4(KO) to identify factors which underlie focal neurodegeneration. Metabolomics revealed that free amino acid concentrations are broadly different by region, and glucose metabolites are increased in a manner dependent on both region and genotype. We then tested the impact of the mTOR inhibitor rapamycin, which dramatically attenuates LS in Ndufs4(KO), on region specific metabolism. Our data revealed that loss of Ndufs4 drives pathogenic changes to CNS glutamine/glutamate/α-ketoglutarate metabolism which are rescued by mTOR inhibition Finally, restriction of the Ndufs4 deletion to pre-synaptic glutamatergic neurons recapitulated the whole-body knockout. Together, our findings are consistent with mTOR inhibition alleviating disease by increasing availability of α-ketoglutarate, which is both an efficient mitochondrial complex I substrate in Ndufs4(KO) and an important metabolite related to neurotransmitter metabolism in glutamatergic neurons.
    Keywords:  Genetics; Leigh syndrome; Metabolism; Mitochondria; Mouse; Rapamycin; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.ymgme.2020.03.007
  5. Metabolites. 2020 Apr 18. pii: E158. [Epub ahead of print]10(4):
    Barbier Saint Hilaire P, Rousseau K, Seyer A, Dechaumet S, Damont A, Junot C, Fenaille F.
      Constant improvements to the Orbitrap mass analyzer, such as acquisition speed, resolution, dynamic range and sensitivity have strengthened its value for the large-scale identification and quantification of metabolites in complex biological matrices. Here, we report the development and optimization of Data Dependent Acquisition (DDA) and Sequential Window Acquisition of all THeoretical fragment ions (SWATH-type) Data Independent Acquisition (DIA) workflows on a high-field Orbitrap FusionTM TribridTM instrument for the robust identification and quantification of metabolites in human plasma. By using a set of 47 exogenous and 72 endogenous molecules, we compared the efficiency and complementarity of both approaches. We exploited the versatility of this mass spectrometer to collect meaningful MS/MS spectra at both high- and low-mass resolution and various low-energy collision-induced dissociation conditions under optimized DDA conditions. We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.
    Keywords:  DDA; DIA; Orbitrap Fusion; high resolution mass spectrometry; metabolomics
    DOI:  https://doi.org/10.3390/metabo10040158
  6. Anal Chem. 2020 Apr 22.
    Yu H, Xing S, Nierves L, Lange PF, Huan T.
      The nonlinear signal response of electrospray ionization (ESI) presents a critical limitation for mass spectrometry (MS)-based quantitative analysis. In the field of metabolomics research, this issue has largely remained unaddressed - MS signal intensities are usually directly used to calculate fold changes for quantitative comparison. In this work, we demonstrate that, due to the nonlinear ESI response, signal intensity ratios of a metabolic feature calculated between two samples may not reflect their real metabolic concentration ratios (i.e., fold-change compression), implicating that conventional fold change calculations directly using MS signal intensities can be misleading. In this regard, we developed a quality control (QC) sample-based signal calibration workflow to overcome the quantitative bias caused by the nonlinear ESI response. In this workflow, calibration curves for every metabolic feature are first established using QC sample injected in serial injection volumes. The MS signals of each metabolic feature are then calibrated to their equivalent QC injection volumes for comparative analysis. We demonstrated this novel workflow in a targeted metabolite analysis, showing that the accuracy of fold change calculations can be significantly improved. Furthermore, in a metabolomic comparison of the bone marrow interstitial fluid samples from leukemia patients before and after chemotherapy, an additional 59 significant metabolic features were found with fold change larger than 1.5 and an additional 97 significant metabolic features had fold changes corrected by more than 0.1. This work enables high-quality quantitative analysis in untargeted metabolomics, thus providing more confident biological hypotheses generation.
    DOI:  https://doi.org/10.1021/acs.analchem.0c00246
  7. Acta Pharm Sin B. 2020 Apr;10(4): 582-602
    Deng H, Li W.
      Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalysing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degradation of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clinical phase 2 studies for neurological disorders and other diseases. This review summarizes the diverse (patho)physiological roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large number of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.
    Keywords:  2-AG, 2-arachidonoyl glycerol; 2-Arachidaoylglycerol; 2-OG, 2-oleoylglycerol; 4-NPA, 4-nitrophenylacetate; 7-HCA, 7-hydroxycoumarinyl arachidonate; AA, arachidonic acid; ABHD6 and ABHD12, α/β-hydrolase 6 and 12; ABP, activity-based probes; ABPP, activity-based protein profiling; AD, Alzheimer's disease; AEA, anandamide; Arachidonic acid; BCRP, breast cancer resistant protein; CB1R and CB2R, cannabinoid receptors; CC-ABPP, click chemistry activity-based protein profiling; CFA, complete Freund's adjuvant; CNS, central nervous system; COX, cyclooxygenases; CYP, cytochrome P450 proteins; Cancer; DAG, diacylglycerol; DAGLs, diacylglycerol lipases; DTT, dithiothreitol; Drug discovery; EAE, encephalomyelitis; EI, enzyme–inhibitor complex; FAAH, amide hydrolase; FFAs, free fatty acids; FP, fluorophosphonate; FP-Rh, fluorophosphonate-rhodamine; FQ, fit quality; HFD, high-fat diet; HFIP, hexafluoroisopropyl; LC–MS, liquid chromatographic mass spectrometry; LFD, low-fat diet; MAGL, monoacylglycerol lipase; MAGs, monoglycerides; MS, multiple sclerosis; Metabolic syndrome; Monoacylglycerol lipases; NAM, N-arachidonoyl maleimide; NHS, N-hydroxysuccinimidyl; Neuroinflammation; OCT2, organic cation transporter 2; P-gp, P-glycoprotein; PA, phosphatidic acid; PD, Parkinson's disease; PET, positron emission tomography; PGE2, prostaglandin; PGs, prostaglandins; PK, pharmacokinetic; PLA2G7, phospholipase A2 group VII; SAR, structure–activity relationship; SBDD, structure-based drug design; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; THL, tetrahydrolipstatin; cPLA2, cytosolic phospholipase A2
    DOI:  https://doi.org/10.1016/j.apsb.2019.10.006
  8. Analyst. 2020 Apr 24.
    Jia M, Peng Z, Yang K, Su C, Wang Y, Yan C.
      Much evidence suggested that cholesterol, eicosanoid and phospholipid metabolism plays crucial roles in inflammation, atherosclerosis, carcinogenesis, etc. Therefore, fast and accurate quantification of the metabolites in these metabolic pathways is necessary for discovering the molecular mechanisms and biomarkers of related diseases. In this assay, ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry platform (UPLC-QqQ-MS) based protocols were developed to simultaneously quantify a total of 104 key metabolites including 32 phospholipids (PLs), 44 eicosanoids (EAs), 28 oxysterols and bile acids (BAs), within 15 minutes. Validation results showed that this method is stable, sensitive and accurate for analyzing different matrix samples. Next, this method was used to characterize the metabolic phenotype of a CCl4-induced liver injury model. The results showed that polyunsaturated fatty acids (PUFA) and PUFA acyl-phospholipids (PFA-PLs) were down-regulated and the levels of saturated fatty acyl-phospholipids (SFA-PLs) and EAs were up-regulated in both the liver tissue and plasma of the CCl4-injury group. BAs were up-regulated in plasma, but down-regulated in the liver tissue of the CCl4-injury group. Immunohistochemistry assay demonstrated that the expression levels of cytosolic phospholipase A2 (cPLA2), phosphorylated cytosolic phospholipase A2 (p-cPLA2), secreted phospholipase A2-IIA (sPLA2-IIA) and lysophosphatidylcholine acyltransferase 1 (LPCAT1) were all up-regulated. According to our results, we drew a diagram of the CCl4-induced acute liver injury molecular mechanism. Moreover, we found that the area under the receiver operating characteristic curve (AUC) of 7α-hydroxycholesterol and 7β-hydroxycholesterol was 1.0, which indicates that the two metabolites have significant potential for the diagnosis of acute liver injury. The outstanding performance of this analytical method proves its further usefulness for mechanism studies and biomarker screening of related diseases.
    DOI:  https://doi.org/10.1039/d0an00385a
  9. Mol Syst Biol. 2020 Apr;16(4): e9247
    Oberhuber M, Pecoraro M, Rusz M, Oberhuber G, Wieselberg M, Haslinger P, Gurnhofer E, Schlederer M, Limberger T, Lagger S, Pencik J, Kodajova P, Högler S, Stockmaier G, Grund-Gröschke S, Aberger F, Bolis M, Theurillat JP, Wiebringhaus R, Weiss T, Haitel A, Brehme M, Wadsak W, Griss J, Mohr T, Hofer A, Jäger A, Pollheimer J, Egger G, Koellensperger G, Mann M, Hantusch B, Kenner L.
      Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.
    Keywords:   OXPHOS ; PDK4; STAT3; TCA cycle; prostate cancer
    DOI:  https://doi.org/10.15252/msb.20199247
  10. Cancers (Basel). 2020 Apr 22. pii: E1031. [Epub ahead of print]12(4):
    Amsalem Z, Arif T, Shteinfer-Kuzmine A, Chalifa-Caspi V, Shoshan-Barmatz V.
      Carcinogenesis is a complicated process that involves the deregulation of epigenetics, resulting in cellular transformational events, such as proliferation, differentiation, and metastasis. Most chromatin-modifying enzymes utilize metabolites as co-factors or substrates and thus are directly dependent on such metabolites as acetyl-coenzyme A, S-adenosylmethionine, and NAD+. Here, we show that using specific siRNA to deplete a tumor of VDAC1 not only led to reprograming of the cancer cell metabolism but also altered several epigenetic-related enzymes and factors. VDAC1, in the outer mitochondrial membrane, controls metabolic cross-talk between the mitochondria and the rest of the cell, thus regulating the metabolic and energetic functions of mitochondria, and has been implicated in apoptotic-relevant events. We previously demonstrated that silencing VDAC1 expression in glioblastoma (GBM) U-87MG cell-derived tumors, resulted in reprogramed metabolism leading to inhibited tumor growth, angiogenesis, epithelial-mesenchymal transition and invasiveness, and elimination of cancer stem cells, while promoting the differentiation of residual tumor cells into neuronal-like cells. These VDAC1 depletion-mediated effects involved alterations in transcription factors regulating signaling pathways associated with cancer hallmarks. As the epigenome is sensitive to cellular metabolism, this study was designed to assess whether depleting VDAC1 affects the metabolism-epigenetics axis. Using DNA microarrays, q-PCR, and specific antibodies, we analyzed the effects of si-VDAC1 treatment of U-87MG-derived tumors on histone modifications and epigenetic-related enzyme expression levels, as well as the methylation and acetylation state, to uncover any alterations in epigenetic properties. Our results demonstrate that metabolic rewiring of GBM via VDAC1 depletion affects epigenetic modifications, and strongly support the presence of an interplay between metabolism and epigenetics.
    Keywords:  VDAC1; cancer; histones epigenetics; metabolism; mitochondria
    DOI:  https://doi.org/10.3390/cancers12041031
  11. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 04. pii: S1570-0232(20)30027-1. [Epub ahead of print]1145 122105
    Folberth J, Begemann K, Jöhren O, Schwaninger M, Othman A.
      As part of the "omics" technologies in the life sciences, metabolomics is becoming increasingly important. In untargeted metabolomics, unambiguous metabolite identification and the inevitable coverage bias that comes with the selection of analytical conditions present major challenges. Reliable compound annotation is essential for translating metabolomics data into meaningful biological information. Here, we developed a fast and transferable method for generating in-house MS2 libraries to improve metabolite identification. Using the new method we established an in-house MS2 library that includes over 4,000 fragmentation spectra of 506 standard compounds for 6 different normalized collision energies (NCEs). Additionally, we generated a comprehensive liquid chromatography (LC) library by testing 57 different LC-MS conditions for 294 compounds. We used the library information to develop an untargeted metabolomics screen with maximum coverage of the metabolome that was successfully tested in a study of 360 human serum samples. The current work demonstrates a workflow for LC-MS/MS-based metabolomics, with enhanced metabolite identification confidence and the possibility to select suitable analysis conditions according to the specific research interest.
    Keywords:  HRMS; Hydrophilic interaction chromatography; In-house library; LC-ESI-MS/MS; Metabolomics; Orbitrap
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122105
  12. J Cancer. 2020 ;11(13): 3944-3954
    Zhu ZJ, Qi Z, Zhang J, Xue WH, Li LF, Shen ZB, Li ZY, Yuan YL, Wang WB, Zhao J.
      Background: Esophageal squamous cell carcinoma (ESCC) is one of the most fatal diseases worldwide. Because early diagnosis is difficult, ESCC is mostly diagnosed at an advanced stage, leading to a poor overall prognosis. The purpose of this study was to explore the differences between plasma metabolic profiles in ESCC patients and healthy controls and to establish a diagnostic model of ESCC. Methods: In this study, a cohort of 310 subjects, containing 140 ESCC patients and 170 healthy controls (HC), was recruited. Participants were randomly separated into a training set (80 ESCCs, 80 HCs) and a validation set (60 ESCCs, 90 HCs) and their plasma metabolomics profiles were analyzed by ultra-performance liquid chromatography-tandem quadruple time-of-flight mass spectrometry (UPLC-QTOF/MS) technique. Univariate statistical analysis and multivariate analysis (MVA) methods were used to identify differential metabolites. Finally, the dysregulated pathways associated with ESCC were further explored and the diagnostic performance of the biomarker panel was evaluated. Results: Metabolic analyses identified 34 significant metabolites involved in the metabolism of amino acids, phospholipids, fatty acids, purine, and choline. Farthermore, an effective diagnostic model for ESCC was constructed based on eight metabolites. This panel of biomarkers consisted of hypoxanthine, proline betaine, indoleacrylic acid, inosine, 9-decenoylcarnitine, tetracosahexaenoic acid, LPE (20:4), and LPC (20:5). The model was verified and evaluated in the validation set. The AUC value of the ROC curve was 0.991(95% CI: 0.981-1.000, CI, Confidence interval), with a sensitivity (SE) of 98.8% and a specificity (SP) of 94.9% for the training set and 0.965(95% CI: 0.936-0.993), with a SE of 88.3% and a SP of 88.9% for the validation set. Among them, three biomarkers, indoleacrylic acid, LPC (20:5), and LPE (20:4), exhibited a trend associated with the ESCC progression. Conclusions: Our study identified a novel plasma biomarker panel, which clearly distinguishes ESCC patients and provides insight into the mechanisms of ESCC. This finding may form the basis for the development of a minimally invasive method for ESCC detection.
    Keywords:  diagnostic model; esophageal squamous cell carcinoma; mass spectrometry; metabolomics; plasma biomarkers
    DOI:  https://doi.org/10.7150/jca.41733
  13. EBioMedicine. 2020 Apr 21. pii: S2352-3964(20)30074-8. [Epub ahead of print]54 102699
    Nwosu ZC, Piorońska W, Battello N, Zimmer AD, Dewidar B, Han M, Pereira S, Blagojevic B, Castven D, Charlestin V, Holenya P, Lochead J, De La Torre C, Gretz N, Sajjakulnukit P, Zhang L, Ward MH, Marquardt JU, di Magliano MP, Lyssiotis CA, Sleeman J, Wölfl S, Ebert MP, Meyer C, Hofmann U, Dooley S.
      BACKGROUND: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed.METHODS: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients.
    FINDINGS: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues.
    INTERPRETATION: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients. FUND: DFG, BMBF and Sino-German Cooperation Project.
    Keywords:  Aerobic glycolysis; Glutamine; HCC; Kinase inhibitors; Metabolic state; Proliferation; Serine
    DOI:  https://doi.org/10.1016/j.ebiom.2020.102699
  14. Biochimie. 2020 Apr 21. pii: S0300-9084(20)30074-2. [Epub ahead of print]
    Rooney M, Bottiglieri T, Wasek-Patterson B, McMahon A, Hughes CF, McCann A, Horigan G, Strain JJ, McNulty H, Ward M.
      Homozygosity for the C677T polymorphism in MTHFR (TT genotype) is associated with a 24-87% increased risk of hypertension. Blood pressure (BP) lowering was previously reported in adults with the TT genotype, in response to supplementation with the MTHFR cofactor, riboflavin. Whether the BP phenotype associated with the polymorphism is related to perturbed one-carbon metabolism is unknown. This study investigated one carbon metabolites and their responsiveness to riboflavin in adults with the TT genotype. Plasma samples from adults (n 115) screened for the MTHFR genotype, who previously participated in RCTs to lower BP, were analysed for methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), betaine, choline and cystathionine by liquid chromatography tandem mass spectrometry (LC-MS/MS). The one-carbon metabolite response to riboflavin (1.6 mg/d; n 24) or placebo (n 23) for 16 weeks in adults with the TT genotype was also investigated. Plasma SAM (74.7 ± 21.0 vs 85.2 ± 22.6 nmol/L, P = 0.013) and SAM:SAH ratio (1.66 ± 0.55 vs 1.85 ± 0.51, P = 0.043) were lower and plasma homocysteine was higher (P = 0.043) in TT, compared to CC individuals. In response to riboflavin, SAM (P = 0.008) and cystathionine (P = 0.045) concentrations increased, with no responses in other one-carbon metabolites. These findings confirm perturbed one-carbon metabolism in individuals with the MTHFR 677 TT genotype, and for the first time demonstrate that SAM, and cystathionine, increase in response to riboflavin supplementation in this genotype group. The genotype-specific, one-carbon metabolite responses to riboflavin intervention observed could offer some insight into the role of this gene-nutrient interaction in blood pressure.
    Keywords:  Hypertension; MTHFR; One-carbon metabolism; Riboflavin; S-adenosylmethionine
    DOI:  https://doi.org/10.1016/j.biochi.2020.04.004
  15. Metabolomics. 2020 Apr 24. 16(5): 59
    Graham SF, Turkoglu O, Yilmaz A, Ustun I, Ugur Z, Bjorndhal T, Han B, Mandal R, Wishart D, Bahado-Singh RO.
      INTRODUCTION: Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by deficiencies in social interactions and communication, combined with restricted and repetitive behavioral issues.OBJECTIVES: As little is known about the etiopathophysiology of ASD and early diagnosis is relatively subjective, we aim to employ a targeted, fully quantitative metabolomics approach to biochemically profile post-mortem human brain with the overall goal of identifying metabolic pathways that may have been perturbed as a result of the disease while uncovering potential central diagnostic biomarkers.
    METHODS: Using a combination of 1H NMR and DI/LC-MS/MS we quantitatively profiled the metabolome of the posterolateral cerebellum from post-mortem human brain harvested from people who suffered with ASD (n = 11) and compared them with age-matched controls (n = 10).
    RESULTS: We accurately identified and quantified 203 metabolites in post-mortem brain extracts and performed a metabolite set enrichment analyses identifying 3 metabolic pathways as significantly perturbed (p < 0.05). These include Pyrimidine, Ubiquinone and Vitamin K metabolism. Further, using a variety of machine-based learning algorithms, we identified a panel of central biomarkers (9-hexadecenoylcarnitine (C16:1) and the phosphatidylcholine PC ae C36:1) capable of discriminating between ASD and controls with an AUC = 0.855 with a sensitivity and specificity equal to 0.80 and 0.818, respectively.
    CONCLUSION: For the first time, we report the use of a multi-platform metabolomics approach to biochemically profile brain from people with ASD and report several metabolic pathways which are perturbed in the diseased brain of ASD sufferers. Further, we identified a panel of biomarkers capable of distinguishing ASD from control brains. We believe that these central biomarkers may be useful for diagnosing ASD in more accessible biomatrices.
    Keywords:  Autism spectrum disorder; Biochemical pathways; Biomarkers; Brain; Machine learning; Metabolomics
    DOI:  https://doi.org/10.1007/s11306-020-01685-z
  16. Anal Bioanal Chem. 2020 Apr 22.
    Robison HM, Chini CE, Comi TJ, Ryu SW, Ognjanovski E, Perry RH.
      In breast cancer, overexpression of human epidermal growth factor receptor 2 (HER2) correlates with overactivation of lipogenesis, mutation of tumor suppressor p53, and increased metastatic potential. The mechanisms through which lipids mediate p53, HER2, and metastatic potential are largely unknown. We have developed a desorption electrospray ionization mass spectrometry (DESI-MS) method to identify lipid biomarkers of HER2/p53 expression, metastatic potential, and disease state (viz. cancer vs. non-cancerous) in monolayer and suspension breast cancer cell cultures (metastatic potential: MCF-7, T-47D, MDA-MB-231; HER2/p53: HCC2218 (HER2+++/p53+), HCC1599 (HER2-/p53-), HCC202 (HER2++/p53-), HCC1419 (HER2+++/p53-) HCC70 (HER2-/p53+++); non-cancerous: MCF-10A). Unsupervised principal component analysis (PCA) of DESI-MS spectra enabled identification of twelve lipid biomarkers of metastatic potential and disease state, as well as ten lipids that distinguish cell lines based on HER2/p53 expression levels (> 200 lipids were identified per cell line). In addition, we developed a DESI-MS imaging (DESI-MSI) method for mapping the spatial distribution of lipids in metastatic spheroids (MDA-MB-231). Of the twelve lipids that correlate with changes in the metastatic potential of monolayer cell cultures, three were localized to the necrotic core of spheroids, indicating a potential role in promoting cancer cell survival in nutrient-deficient environments. One lipid species, which was not detected in monolayer MDA-MB-231 cultures, was spatially localized to the periphery of the spheroid, suggesting a potential role in invasion and/or proliferation. These results demonstrate that combining DESI-MS/PCA of monolayer and suspension cell cultures with DESI-MSI of spheroids is a promising approach for identifying lipid biomarkers of specific genotypes and phenotypes, as well as elucidating the potential function of these biomarkers in breast cancer. Graphical Absract.
    Keywords:  Breast cancer; Desorption electrospray ionization; Lipid; Mass spectrometry imaging; Spheroid
    DOI:  https://doi.org/10.1007/s00216-020-02537-4
  17. Front Med (Lausanne). 2020 ;7 113
    Pant K, Richard S, Peixoto E, Gradilone SA.
      Cholangiocarcinoma (CCA) is one of the most lethal cancers, and its rate of occurrence is increasing annually. The diagnoses of CCA patients remain elusive due to the lack of early symptoms and is misdiagnosed as HCC in a considerable percentage of patients. It is crucial to explore the underlying mechanisms of CCA carcinogenesis and development to find out specific biomarkers for early diagnosis of CCA and new promising therapeutic targets. In recent times, the reprogramming of tumor cells metabolism has been recognized as a hallmark of cancer. The modification from the oxidative phosphorylation metabolic pathway to the glycolysis pathway in CCA meets the demands of cancer cell proliferation and provides a favorable environment for tumor development. The alteration of metabolic programming in cancer cells is complex and may occur via mutations and epigenetic modifications within oncogenes, tumor suppressor genes, signaling pathways, and glycolytic enzymes. Herein we review the altered metabolism in cancer and the signaling pathways involved in this phenomena as they may affect CCA development. Understanding the regulatory pathways of glucose metabolism such as Akt/mTOR, HIF1α, and cMyc in CCA may further develop our knowledge of this devastating disease and may offer relevant information in the exploration of new diagnostic biomarkers and targeted therapeutic approaches for CCA.
    Keywords:  aerobic glycolysis; cholangiocarcinoma; glucose metabolism; metabolic reprogramming; warburg effect
    DOI:  https://doi.org/10.3389/fmed.2020.00113
  18. Nutrients. 2020 Apr 17. pii: E1116. [Epub ahead of print]12(4):
    Xu Y, Zhao T, Ren H, Xie Y, An J, Shang J, Tabys D, Liu N.
      Lactoferrin (LF) exerts a promoting bone health function. The effects of LF on bone formation at the metabolic level have been less explored. Urinary metabolic profiling of growing Sprague-Dawley (SD) rats LF-supplemented (1000 mg/kg bw) for four weeks were explored by Liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum markers of bone formation and bone resorption, the bone mass, and the osteogenesis markers of femur were measured by an enzyme-linked immunosorbent assay, micro-computerized tomography, and immunohistochemistry, respectively. Compared with the control, LF supplementation improved bone formation (p < 0.05), reduced bone resorption (p < 0.05), enhanced femoral bone mineral density and microarchitecture (p < 0.05), and upregulated osteocalcin, osterix, and Runx-2 expression (p < 0.05) of femur. LF upregulated 69 urinary metabolites. KEGG and pathway enrichment analyses of those urinary metabolites, and the Person's correlation analyses among those urinary metabolites and bone status revealed that LF impacted on bone formation via regulatory comprehensive pathways including taurine and hypotaurine metabolism, arginine and proline metabolism, cyanoamino acid metabolism, nitrogen metabolism, nicotinate and nicotinamide metabolism, and fatty acid biosynthesis. The present study indicated the metabolomics is a useful and practical tool to elucidate the mechanisms by which LF augments bone mass formation in growing animals.
    Keywords:  LC-MS/MS; bone formation; lactoferrin; metabolomics; osteogenesis
    DOI:  https://doi.org/10.3390/nu12041116
  19. Nutr Res Rev. 2020 Apr 20. 1-44
    Ulug E, Nergiz-Unal R.
      Currently, prevention and treatment of cardiovascular diseases have been on a global focus since it is the number one cause of mortality and morbidity. In the pathogenesis of cardiovascular diseases, it was generally thought that impaired cholesterol homeostasis might be a risk factor. Cholesterol homeostasis is affected by exogenous factors (i.e. diet) and endogenous factors (i.e. certain receptors, enzymes, and transcription factors). In this context, the number of studies investigating the potential mechanisms of dietary fatty acids on cholesterol homeostasis have increased in recent years. As well, cluster of differentiation 36 (CD36) receptor is multifunctional membrane receptor involved in fatty acids uptake, and lipid metabolism, atherothrombosis, and inflammation. The CD36 proposed to be a crucial molecule in cholesterol homeostasis in various mechanisms including absorption/reabsorption, synthesis, and transport of cholesterol and bile acids. Moreover, it was reported that the amount of fatty acids and fatty acid pattern of the diet influenced the CD36 level and CD36 mediated cholesterol metabolism principally in liver, intestine, and macrophages. In these processes, CD36 mediated cholesterol and lipoprotein homeostasis might be impaired from dietary saturated fatty acids (SFA) and trans fatty acids (TFA), whereas ameliorated from monounsaturated fatty acids (MUFA) in diet. The effects of polyunsaturated fatty acids (PUFA) on CD36 mediated cholesterol homeostasis is controversial depending on the amount of n-3 PUFA, n-6 PUFA, and the ratio of n-3/n-6 PUFA. Thus, since CD36 receptor suggested to be a novel nutrient-sensitive biomarker, role of CD36 and dietary fatty acids on cholesterol metabolism might be considered in medical nutrition therapy in near future. Therefore, the novel nutritional target of CD36 and interventions that focus on dietary fatty acids on potential mechanisms underlying cholesterol homeostasis is discussed in this review.
    Keywords:  CD36; Nutrition; cholesterol metabolism; fatty acids
    DOI:  https://doi.org/10.1017/S0954422420000128
  20. Breast Cancer Res. 2020 Apr 22. 22(1): 38
    Ben Hassen C, Gutierrez-Pajares JL, Guimaraes C, Guibon R, Pinault M, Fromont G, Frank PG.
      BACKGROUND: The highest incidence of breast cancer is in the Western world. Several aspects of the Western lifestyle are known risk factors for breast cancer. In particular, previous studies have shown that cholesterol levels can play an important role in the regulation of tumor progression.METHODS: In the present study, we modulated cholesterol metabolism in the human breast cancer cell lines MCF-7 and MDA-MB-231 using a genetic approach. Apolipoprotein A-I (apoA-I) and apolipoprotein E (apoE) were expressed in these cell lines to modulate cholesterol metabolism. The effects of these apolipoproteins on cancer cell properties were examined.
    RESULTS: Our results show that both apolipoproteins can regulate cholesterol metabolism and can control the epithelial-to-mesenchymal transition process. However, these effects were different depending on the cell type. We show that expressing apoA-I or apoE stimulates proliferation, migration, and tumor growth of MCF-7 cells. However, apoA-I or apoE reduces proliferation and migration of MDA-MB-231 cells.
    CONCLUSIONS: These data suggest that modulating sterol metabolism may be most effective at limiting tumor progression in models of triple-negative cancers.
    Keywords:  Cancer progression; Cholesterol; Lipids
    DOI:  https://doi.org/10.1186/s13058-020-01276-9
  21. Rapid Commun Mass Spectrom. 2020 Apr 19. e8814
    Tokuoka SM, Yasumoto A, Kita Y, Shimizu T, Yatomi Y, Oda Y.
      RATIONALE: Electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, the normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard material (IS) labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values.METHODS: Triple quadrupole MS coupled with liquid chromatography (LC) was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories.
    RESULTS: Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since the ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard method, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available.
    CONCLUSIONS: Our results raise the question whether the quantitative method using stable-isotope-labeled IS labeled is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined by ESI-MS and only highly substituted deuterium labeled IS was used.
    DOI:  https://doi.org/10.1002/rcm.8814
  22. Anal Chem. 2020 Apr 20.
    Matsuoka Y, Izumi Y, Takahashi M, Bamba T, Yamada KI.
      Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we pro-posed a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed, for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.
    DOI:  https://doi.org/10.1021/acs.analchem.0c00053
  23. Anal Chem. 2020 Apr 23.
    Dazzoni R, Buré C, Morvan E, Grélard A, Gounou C, Schmitter JM, Loquet A, Larijani B, Dufourc EJ.
      The human nuclear membrane is composed of a double bilayer; the inner membrane being linked to the protein lamina net-work and the outer nuclear membrane continuous with the endoplasmic reticulum. Nuclear membranes can form large invag-inations inside the nucleus whose role are poorly known. Although most of the protein identification has been determined, their lipid composition remains largely undetermined. In order to understand the mechanical and dynamic properties of nu-clear membranes we investigated their lipid composition by two quantitative methods, 31P and 1H multidimensional NMR and mass spectrometry using internal standards. We also developed a non-detergent nuclei extraction protocol allowing to produce milligram quantities of nuclear membrane lipids. We found that the nuclear membrane lipid extract is composed of a complex mixture of phospholipids with phosphatidylcholines present in large amounts. Negatively charged lipids, with high amounts of phosphoinositides were also present. Mass spectrometry confirmed the phospholipid composition and provided further information on acyl-chain length and unsaturation. Lipid chain lengths range between 30 to 38 carbon atoms (two chains summed up) with a high proportion of 34 carbon atom length for most species. PI lipids have large amounts of chain length with 36-38 carbons. Independent of the chain length unsaturations were highly elevated with 1 to 2 double bonds per lipid species.
    DOI:  https://doi.org/10.1021/acs.analchem.9b05052
  24. Acta Physiol (Oxf). 2020 Apr 24.
    Sieckmann T, Kirschner KM.
      Metabolomics is now, after genomics, transcriptomics, and proteomics, one of the -omics that is beginning to expand into clinical diagnostics. This progress has been achieved by the recent development of high throughput screening methods. We are all familiar with metabolites like sugars, amino acids, lipids or nucleotides. But there are many more of these small molecules. Metabolites include all intermediates and products of cellular metabolism. Metabolome refers to the whole collection of metabolites in a biological system.
    DOI:  https://doi.org/10.1111/apha.13480
  25. Expert Rev Proteomics. 2020 Apr 20.
    Liu T, Tan Z, Yu J, Feng P, Guo J, Meng W, Chen Y, Rao T, Liu Z, Peng J.
      Background: Colorectal cancer (CRC) represents a third leading cause of cancer-related death worldwide. The reliable diagnostic biomarkers for detecting CRC at early stage is critical for decreasing the mortality.Method: A conjunctive lipidomic approach was employed to investigate the differences in plasma lipid profiles of CRC patients (n = 101) and healthy volunteers (n = 52). Based on UHPLC-Q-TOF MS and UHPLC-QQQ MS platforms, a total of 235 lipids were structurally detected. Multivariate data analysis was conducted including partial least squared discriminant analysis (PLS-DA), fold change performance and Mann-Whitney U test.Results: A total of 11 lipid species, including 1 Glycerophosphoethanolamine (PE), 3 ethanolamine plasmalogens (PlsEtn), 1 plasmanyl glycerophosphatidylethanolamine (PE-O), 3 fatty acids (FFA), 1 Fatty acid ester of hydroxyl fatty acid (FAHFA), and 2 Diacylglycerophosphates (PA) were identified to distinguish the CRC patients at early stage from healthy controls. In addition, these potential lipid biomarkers achieved an estimated AUC=0.981 in a validation set for univariate ROC analysis.Conclusion: By combining Q-TOF MS and QQQ MS analysis, the 11 lipids exhibited good performance in differentiating early-stage CRC and healthy control. This study also demonstrated that the lipidomics is a powerful tool in discovering new potential biomarkers for cancer diagnosis.
    Keywords:  Q-TOF MS; QQQ MS; colorectal cancer; diagnostic biomarkers; lipidomics
    DOI:  https://doi.org/10.1080/14789450.2020.1757443
  26. Cell Death Dis. 2020 Apr 23. 11(4): 267
    Gong J, Lin Y, Zhang H, Liu C, Cheng Z, Yang X, Zhang J, Xiao Y, Sang N, Qian X, Wang L, Cen X, Du X, Zhao Y.
      Metabolic interaction between cancer-associated fibroblasts (CAFs) and colorectal cancer (CRC) cells plays a major role in CRC progression. However, little is known about lipid alternations in CAFs and how these metabolic reprogramming affect CRC cells metastasis. Here, we uncover CAFs conditioned medium (CM) promote the migration of CRC cells compared with normal fibroblasts CM. CAFs undergo a lipidomic reprogramming, and accumulate more fatty acids and phospholipids. CAFs CM after protein deprivation still increase the CRC cells migration, which suggests small molecular metabolites in CAFs CM are responsible for CRC cells migration. Then, we confirm that CRC cells take up the lipids metabolites that are secreted from CAFs. Fatty acids synthase (FASN), a crucial enzyme in fatty acids synthesis, is significantly increased in CAFs. CAF-induced CRC cell migration is abolished by knockdown of FASN by siRNA or reducing the uptake of fatty acids by CRC cells by sulfo-N-succinimidyloleate sodium in vitro and CD36 monoclonal antibody in vivo. To conclude, our results provide a new insight into the mechanism of CRC metastasis and suggest FASN of CAFs or CD36 of CRC cells may be potential targets for anti-metastasis treatment in the future.
    DOI:  https://doi.org/10.1038/s41419-020-2434-z
  27. Kidney Dis (Basel). 2020 Mar;6(2): 125-134
    Chen Y, Wen P, Yang J, Niu J.
      Background: Key pathogenetic mechanisms underlying renal disease progression are unaffected by current treatment. Metabolite profiling has significantly contributed to a deeper understanding of the biochemical metabolic networks and pathways in disease, but the biochemical details in maintenance hemodialysis (MHD) patients remain largely undefined.Methods: The metabolic fingerprinting of plasma samples from 19 MHD patients and 12 healthy controls was characterized using liquid chromatography quadrupole time-of-flight mass spectrometry. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to analyze the metabolic data.
    Results: The plasma metabolite profile distinguished the MHD patients from the healthy controls successfully by using both PCA and OPLS-DA models. Sixty-three metabolites were identified as the key metabolites to discriminate the MHD patients from healthy controls, involving several metabolic pathways (all p < 0.05). An increase in plasma levels of D-glucose, hippuric acid, androsterone glucuronide, indolelactic acid, and a reduction in plasma levels of glycerophosphocholine, serotonin, L-lactic acid, phytosphingosine, and several lysophosphatidylcholine were observed in MHD patients compared to healthy subjects. Metabolomics analysis combined with KEGG pathway enrichment analysis revealed that non-alcoholic fatty liver disease, choline metabolism in cancer, the forkhead box O signaling pathway, and the hypoxia-inducible factor-1 signaling pathway in MHD patients were significantly changed (p < 0.05).
    Conclusion: The identification of a novel signaling pathway and key metabolite markers in MHD patients provides insights into potential pathogenesis and valuable pharmacological targets for end-stage renal disease.
    Keywords:  End-stage renal disease; Liquid chromatography quadrupole time-of-flight mass spectrometry; Metabolic profiling
    DOI:  https://doi.org/10.1159/000505156
  28. Int J Oncol. 2020 Apr 08.
    Liao S, Liang L, Yue C, He J, He Z, Jin X, Luo G, Zhou Y.
      In contrast to normal cells, cancer cells typically undergo metabolic reprogramming. Studies have shown that oncogenes play an important role in this metabolic reprogramming. CD38 is a multifunctional transmembrane protein that is expressed abnormally in a variety of tumor types. To investigate the effect and possible mechanism of CD38 in cervical cancer cells and to provide a new therapeutic target for the treatment of cervical cancer, the present study identified that CD38 is involved in regulating cell metabolism in cervical cancer cells. Liquid chromatography‑tandem mass spectrometry and bioinformatic analyses revealed that differentially abundant proteins in CD38‑overexpressed cervical cancer cells (CaSki‑CD38 and HeLa‑CD38) are predominantly involved in glycolytic pathways, oxidative phosphorylation and the NAD/NADH metabolic process. Further experiments using an ATP test kit and lactate test kit revealed that CD38 promotes glucose consumption, increases lactate accumulation and increases ATP production. In addition, CD38 increases the phosphorylation of phosphatidylserine/threonine kinase (AKT), mechanistic target of rapamycin (mTOR) and phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K), which play a key role in tumor metabolism. Furthermore, it was found that the energy metabolism of cervical cancer cells was inhibited following treatment with the mTOR inhibitor rapamycin. In conclusion, the results of the present study suggested that CD38 regulates the metabolism of cervical cancer cells by regulating the PI3K/AKT/mTOR pathway, which may be a candidate target for the treatment of cervical cancer.
    DOI:  https://doi.org/10.3892/ijo.2020.5040
  29. Curr Opin Biotechnol. 2020 Apr 15. pii: S0958-1669(20)30035-5. [Epub ahead of print]64 151-160
    Xu J, Martien J, Gilbertson C, Ma J, Amador-Noguez D, Park JO.
      Metabolite concentrations, fluxes, and free energies constitute the basis for understanding and controlling metabolism. Mass spectrometry and stable isotopes are integral tools in quantifying these metabolic features. For absolute metabolite concentration and flux measurement, 13C internal standards and tracers have been the gold standard. In contrast, no established methods exist for comprehensive thermodynamic quantitation under physiological environments. Recently, using high-resolution mass spectrometry and multi-isotope tracing, flux quantitation has been increasingly adopted in broader metabolism. The improved flux quantitation led to determination of Gibbs free energy of reaction (ΔG) in central carbon metabolism using a relationship between reaction reversibility and thermodynamic driving force. Here we highlight recent advances in multi-isotope tracing for metabolic flux and free energy analysis.
    DOI:  https://doi.org/10.1016/j.copbio.2020.02.018
  30. Metabolites. 2020 Apr 17. pii: E156. [Epub ahead of print]10(4):
    Kuang E, Marney M, Cuevas D, Edwards RA, Forsberg EM.
      Genomics-based metabolic models of microorganisms currently have no easy way of corroborating predicted biomass with the actual metabolites being produced. This study uses untargeted mass spectrometry-based metabolomics data to generate a list of accurate metabolite masses produced from the human commensal bacteria Citrobacter sedlakii grown in the presence of a simple glucose carbon source. A genomics-based flux balance metabolic model of this bacterium was previously generated using the bioinformatics tool PyFBA and phenotypic growth curve data. The high-resolution mass spectrometry data obtained through timed metabolic extractions were integrated with the predicted metabolic model through a program called MS_FBA. This program correlated untargeted metabolomics features from C. sedlakii with 218 of the 699 metabolites in the model using an exact mass match, with 51 metabolites further confirmed using predicted isotope ratios. Over 1400 metabolites were matched with additional metabolites in the ModelSEED database, indicating the need to incorporate more specific gene annotations into the predictive model through metabolomics-guided gap filling.
    Keywords:  bioinformatics; flux balance analysis; mass spectrometry; metabolomics; microbiome; multiomics
    DOI:  https://doi.org/10.3390/metabo10040156
  31. Mol Divers. 2020 Apr 24.
    Fragoso-Vázquez MJ, Méndez-Luna D, Rosales-Hernández MC, Luna-Palencia GR, Estrada-Pérez A, Fromager B, Vásquez-Moctezuma I, Correa-Basurto J.
      Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.
    Keywords:  Anti-proliferative LC–MS-based metabolomic; Dual-target inhibitor; Gln-VPA; Glutaminase
    DOI:  https://doi.org/10.1007/s11030-020-10089-z
  32. Cell Death Dis. 2020 Apr 20. 11(4): 253
    Kant S, Kesarwani P, Prabhu A, Graham SF, Buelow KL, Nakano I, Chinnaiyan P.
      Despite advances in molecularly characterizing glioblastoma (GBM), metabolic alterations driving its aggressive phenotype are only beginning to be recognized. Integrative cross-platform analysis coupling global metabolomic and gene expression profiling on patient-derived glioma identified fatty acid β-oxidation (FAO) as a metabolic node in GBM. We determined that the biologic consequence of enhanced FAO is directly dependent upon tumor microenvironment. FAO serves as a metabolic cue to drive proliferation in a β-HB/GPR109A dependent autocrine manner in nutrient favorable conditions, while providing an efficient, alternate source of ATP only in nutrient unfavorable conditions. Rational combinatorial strategies designed to target these dynamic roles FAO plays in gliomagenesis resulted in necroptosis-mediated metabolic synthetic lethality in GBM. In summary, we identified FAO as a dominant metabolic node in GBM that provides metabolic plasticity, allowing these cells to adapt to their dynamic microenvironment. Combinatorial strategies designed to target these diverse roles FAO plays in gliomagenesis offers therapeutic potential in GBM.
    DOI:  https://doi.org/10.1038/s41419-020-2449-5
  33. Mol Omics. 2020 Apr 20.
    Sethi MK, Downs M, Zaia J.
      Advancement in mass spectrometry has revolutionized the field of proteomics. However, there remains a gap in the analysis of protein post-translational modifications (PTMs), particularly for glycosylation. Glycosylation, the most common form of PTM, is involved in most biological processes; thus, analysis of glycans along with proteins is crucial to answering important biologically relevant questions. Of particular interest is the brain extracellular matrix (ECM), which has been called the "final Frontier" in neuroscience, which consists of highly glycosylated proteins. Among these, proteoglycans (PGs) contain large glycan structures called glycosaminoglycans (GAGs) that form crucial ECM components, including perineuronal nets (PNNs), shown to be altered in neuropsychiatric diseases. Thus, there is a growing need for high-throughput methods that combine GAG (glycomics) and PGs (proteomics) analysis to unravel the complete biological picture. The protocol presented here integrates glycomics and proteomics to analyze multiple classes of biomolecules. We use a filter-aided sample preparation (FASP) type serial in-solution digestion of GAG classes, including hyaluronan (HA), chondroitin sulfate (CS), and heparan sulfate (HS), followed by peptides. The GAGs and peptides are then cleaned and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is an efficient and economical way of processing tissue or cell lysates to isolate various GAG classes and peptides from the same sample. The method is more efficient (single-pot) than available parallel (multi-pot) release methods, and removal of GAGs facilitates the identification of the proteins with higher peptide-coverage than using conventional-proteomics. Overall, we demonstrate a high-throughput & efficient protocol for mass spectrometry-based glycomic and proteomic analysis (data are available via ProteomeXchange with identifier PXD017513).
    DOI:  https://doi.org/10.1039/d0mo00019a
  34. J Enzyme Inhib Med Chem. 2020 Dec;35(1): 963-973
    Urbanelli L, Buratta S, Logozzi M, Mitro N, Sagini K, Raimo RD, Caruso D, Fais S, Emiliani C.
      Cancer cells need to modulate the biosynthesis of membrane lipids and fatty acids to adapt themselves to an accelerated rate of cell division and survive into an extracellular environment characterised by a low pH. To gain insight this crucial survival process, we investigated the lipid composition of Mel 501 melanoma cells cultured at either physiological or acidic pH and observed the remodelling of phospholipids towards longer and more unsaturated acyl chains at low pH. This modification was related to changes in gene expression profile, as we observed an up-regulation of genes involved in acyl chain desaturation, elongation and transfer to phospholipids. PC3 prostate and MCF7 breast cancer cells adapted at acidic pH also demonstrated phospholipid fatty acid remodelling related to gene expression changes. Overall findings clearly indicate that low extracellular pH impresses a specific lipid signature to cells, associated with transcriptional reprogramming.
    Keywords:  Phospholipid remodelling; desaturases; elongases; tumour microenvironment; tumour pH
    DOI:  https://doi.org/10.1080/14756366.2020.1748025
  35. J Clin Invest. 2020 Apr 23. pii: 131859. [Epub ahead of print]
    Oh MH, Sun IH, Zhao L, Leone RD, Sun IM, Xu W, Collins SL, Tam AJ, Blosser RL, Patel CH, Englert JM, Arwood ML, Wen J, Chan-Li Y, Tenora L, Majer P, Rais R, Slusher BS, Horton MR, Powell JD.
      Myeloid cells comprise a major component of the tumor-microenvironment (TME) promoting tumor growth and immune evasion. By employing a novel small molecule inhibitor of glutamine metabolism, not only were we able to inhibit tumor growth, but we markedly inhibited the generation and recruitment of myeloid-derived suppressor cells (MDSCs). Targeting tumor glutamine metabolism led to a decrease in CSF3 and hence recruitment of MDSCs as well immunogenic cell death leading to an increase in inflammatory tumor-associated macrophages (TAMs). Alternatively, inhibiting glutamine metabolism of the MDSCs themselves led to activation induced cell death and conversion of MDSCs to inflammatory macrophages. Surprisingly, blocking glutamine metabolism also inhibited IDO expression of both the tumor and myeloid derived cells leading to a marked decrease in kynurenine levels. This in turn inhibited the development of metastasis and further enhanced anti-tumor immunity. Indeed, targeting glutamine metabolism rendered checkpoint blockade-resistant tumors susceptible to immunotherapy. Overall, our studies define an intimate interplay between the unique metabolism of tumors and the metabolism of suppressive immune cells.
    Keywords:  Cancer immunotherapy; Immunology; Innate immunity; Oncology
    DOI:  https://doi.org/10.1172/JCI131859
  36. Anal Bioanal Chem. 2020 Apr 24.
    Xuan Q, Zheng F, Yu D, Ouyang Y, Zhao X, Hu C, Xu G.
      Lipidomics aims to characterize lipid alteration in response to internal or external subtle perturbations in complex biological samples. Lipid abnormality is a major risk factor for many diseases. Large-scale lipidomic studies may offer new insights into the pathophysiological mechanisms of diseases, new opportunities in systems biology, functional biology, and personalized medicine. To this end, a highly efficient and stable lipidomic method is highly in demand. We herein present a rapid and relatively high coverage lipidomic profiling approach based on ultra-high performance liquid chromatography-mass spectrometry by comparing the performance of different chromatographic columns, optimizing the elution gradient and selecting an appropriate data acquisition mode of mass spectra. As a result, a total of 481 lipids were detected from 40 μL serum sample within 13 min, covering 20 common lipid (sub)classes. The developed method was well validated with satisfactory analytical characteristics in linearity, repeatability, stability, and lipid coverage. To show the usefulness, the method was employed to investigate serum lipid profiling of 43 subjects with mild diabetic retinopathy and 44 normal controls, and successfully defined the differential lipids related to diabetic retinopathy. We believe that this rapid method will be beneficial for lipidomic analysis of large-scale clinical samples.
    Keywords:  Diabetic retinopathy; Differential lipids; Lipidome; Lipidomic profiling; Liquid chromatography–mass spectrometry
    DOI:  https://doi.org/10.1007/s00216-020-02632-6
  37. Mol Cell Proteomics. 2020 Apr 20. pii: mcp.P119.001913. [Epub ahead of print]
    Pino LK, Just SC, MacCoss MJ, Searle BC.
      Data independent acquisition (DIA) is an attractive alternative to standard shotgun proteomics methods for quantitative experiments. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent acquisition (DDA) to detect and quantify peptides. In addition to working with non-human samples, studies of splice junctions, sequence variants, or simply working with small sample yields can make developing DDA-based spectrum libraries impractical. Here we illustrate how to acquire, queue, and validate DIA data without spectrum libraries, and provide a workflow to efficiently generate DIA-only chromatogram libraries using gas-phase fractionation (GPF). We present best-practice methods for collecting DIA data using Orbitrap-based instruments, and develop an understanding for why DIA using an Orbitrap mass spectrometer should be approached differently than when using time-of-flight instruments. Finally, we discuss several methods for analyzing DIA data without libraries.
    Keywords:  DIA; Data Independent Acquisition; Data evaluation; Label-free quantification; Mass Spectrometry; Protein Identification*; Quantification
    DOI:  https://doi.org/10.1074/mcp.P119.001913
  38. Cancer Metastasis Rev. 2020 Apr 20.
    Skotland T, Kavaliauskiene S, Sandvig K.
      Several studies have demonstrated interactions between the two leaflets in membrane bilayers and the importance of specific lipid species for such interaction and membrane function. We here discuss these investigations with a focus on the sphingolipid and cholesterol-rich lipid membrane domains called lipid rafts, including the small flask-shaped invaginations called caveolae, and the importance of such membrane structures in cell biology and cancer. We discuss the possible interactions between the very long-chain sphingolipids in the outer leaflet of the plasma membrane and the phosphatidylserine species PS 18:0/18:1 in the inner leaflet and the importance of cholesterol for such interactions. We challenge the view that lipid rafts contain a large fraction of lipids with two saturated fatty acyl groups and argue that it is important in future studies of membrane models to use asymmetric membrane bilayers with lipid species commonly found in cellular membranes. We also discuss the need for more quantitative lipidomic studies in order to understand membrane function and structure in general, and the importance of lipid rafts in biological systems. Finally, we discuss cancer-related changes in lipid rafts and lipid composition, with a special focus on changes in glycosphingolipids and the possibility of using lipid therapy for cancer treatment.
    Keywords:  Cancer; Caveolae; Endocytosis; Leaflet interdigitation; Membrane domains; Molecular dynamic simulation
    DOI:  https://doi.org/10.1007/s10555-020-09872-z
  39. J Cell Biol. 2020 Jun 01. pii: e201906204. [Epub ahead of print]219(6):
    Taskinen ME, Närvä E, Conway JRW, Hinojosa LS, Lilla S, Mai A, De Franceschi N, Elo LL, Grosse R, Zanivan S, Norman JC, Ivaska J.
      Microtubule-associated serine/threonine-protein kinase-like (MASTL) is a mitosis-accelerating kinase with emerging roles in cancer progression. However, possible cell cycle-independent mechanisms behind its oncogenicity remain ambiguous. Here, we identify MASTL as an activator of cell contractility and MRTF-A/SRF (myocardin-related transcription factor A/serum response factor) signaling. Depletion of MASTL increased cell spreading while reducing contractile actin stress fibers in normal and breast cancer cells and strongly impairing breast cancer cell motility and invasion. Transcriptome and proteome profiling revealed MASTL-regulated genes implicated in cell movement and actomyosin contraction, including Rho guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and MRTF-A target genes tropomyosin 4.2 (TPM4), vinculin (VCL), and nonmuscle myosin IIB (NM-2B, MYH10). Mechanistically, MASTL associated with MRTF-A and increased its nuclear retention and transcriptional activity. Importantly, MASTL kinase activity was not required for regulation of cell spreading or MRTF-A/SRF transcriptional activity. Taken together, we present a previously unknown kinase-independent role for MASTL as a regulator of cell adhesion, contractility, and MRTF-A/SRF activity.
    DOI:  https://doi.org/10.1083/jcb.201906204
  40. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 03. pii: S1570-0232(20)30024-6. [Epub ahead of print]1145 122099
    Kotapati HK, Bates PD.
      Three normal phase HPLC methods were produced to separate lipid classes on a PVA-Sil stationary phase including: 9 polar lipids (method 1); 13 combined polar and neutral lipids (method 2); and a combined method that further separates the neutral lipids into 2-4 subclasses based on the presence of fatty acids containing a polar functional group (e.g. hydroxyl) for a total of 20 lipid classes and subclasses separated in a single run (method 3). Polar lipids separated include: the phosphoglycerolipids PG, PE, PI, PS, PC and LPC; the galactoglycerolipids MGDG and DGDG; and a sulfoglycerolipid SQDG. Neutral lipids include TAG, DAG, and MAG classes and sub-classes containing 0-3, 0-2, and 0-1 hydroxy fatty acids, respectively. The hexane/isopropanol/methanol/aqueous system separates polar lipids without the use of chloroform such that it is suitable for radioactivity analysis by in-line flow scintillation counting. Each method was optimized using the natural lipid standards comprised of diverse molecular species that were detected by ELSD. All molecular species of each lipid class eluted together as single peak detected by ELSD. The methods were demonstrated to be suitable for resolving lipid extracts from animal, microbial, and plant sources as well as application to 14C based metabolic tracing of lipid metabolism in leaves and seeds.
    Keywords:  Galactolipids; Hydroxy fatty acids; Metabolic flux; Phospholipids; Radioactivity; Triglycerides
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122099
  41. Int J Mol Sci. 2020 Apr 20. pii: E2873. [Epub ahead of print]21(8):
    Chen C, Hou J, Tanner JJ, Cheng J.
      Recent advances in mass spectrometry (MS)-based proteomics have enabled tremendous progress in the understanding of cellular mechanisms, disease progression, and the relationship between genotype and phenotype. Though many popular bioinformatics methods in proteomics are derived from other omics studies, novel analysis strategies are required to deal with the unique characteristics of proteomics data. In this review, we discuss the current developments in the bioinformatics methods used in proteomics and how they facilitate the mechanistic understanding of biological processes. We first introduce bioinformatics software and tools designed for mass spectrometry-based protein identification and quantification, and then we review the different statistical and machine learning methods that have been developed to perform comprehensive analysis in proteomics studies. We conclude with a discussion of how quantitative protein data can be used to reconstruct protein interactions and signaling networks.
    Keywords:  bioinformatics analysis; computational proteomics; machine learning
    DOI:  https://doi.org/10.3390/ijms21082873
  42. Nat Commun. 2020 Apr 20. 11(1): 1891
    Roumans KHM, Lindeboom L, Veeraiah P, Remie CME, Phielix E, Havekes B, Bruls YMH, Brouwers MCGJ, Ståhlman M, Alssema M, Peters HPF, de Mutsert R, Staels B, Taskinen MR, Borén J, Schrauwen P, Schrauwen-Hinderling VB.
      Hepatic steatosis is associated with poor cardiometabolic health, with de novo lipogenesis (DNL) contributing to hepatic steatosis and subsequent insulin resistance. Hepatic saturated fatty acids (SFA) may be a marker of DNL and are suggested to be most detrimental in contributing to insulin resistance. Here, we show in a cross-sectional study design (ClinicalTrials.gov ID: NCT03211299) that we are able to distinguish the fractions of hepatic SFA, mono- and polyunsaturated fatty acids in healthy and metabolically compromised volunteers using proton magnetic resonance spectroscopy (1H-MRS). DNL is positively associated with SFA fraction and is elevated in patients with non-alcoholic fatty liver and type 2 diabetes. Intriguingly, SFA fraction shows a strong, negative correlation with hepatic insulin sensitivity. Our results show that the hepatic lipid composition, as determined by our 1H-MRS methodology, is a measure of DNL and suggest that specifically the SFA fraction may hamper hepatic insulin sensitivity.
    DOI:  https://doi.org/10.1038/s41467-020-15684-0