bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒07‒05
twenty-six papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research


  1. Methods Mol Biol. 2020 ;2156 203-239
    Erban A, Martinez-Seidel F, Rajarathinam Y, Dethloff F, Orf I, Fehrle I, Alpers J, Beine-Golovchuk O, Kopka J.
      This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of plant material that was exposed to temperature stress. The method can be combined with stable isotope labeling and tracing experiments and is equally applicable to preparations of plant material and microbial photosynthetic organisms. The described methods are modular and can be multiplexed, that is, the same sample or a paired identical backup sample can be analyzed sequentially by more than one of the described procedures. The modules include rapid sampling and metabolic inactivation protocols for samples in a wide weight range, sample extraction procedures, chemical derivatization steps that are required to make the metabolite fraction amenable to gas chromatographic analysis, routine GC-MS methods, and procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The methods have two applications: (1) The rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification by exact quantification using GC-MS protocols that are extended by internal and external standardization.
    Keywords:  Absolute quantification; GC-MS; Gas chromatography; Metabolism; Metabolite profiling; Metabolomics; Relative quantification; Stable isotope labeling; TOF-MS; Time-of-flight mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-0660-5_15
  2. Anal Chem. 2020 Jul 02.
    Lu W, Xing X, Wang L, Chen L, Zhang S, McReynolds MR, Rabinowitz JD.
      Annotation of untargeted high-resolution full-scan LC-MS metabolomics data remains challenging due to individual metabolites generating multiple LC-MS peaks arising from isotopes, adducts and fragments. Adduct annotation is a particular challenge, as the same mass difference between peaks can arise from adduct formation, fragmentation, or different biological species. To address this, here we describe a Buffer Modification Workflow (BMW), in which the same sample is run by LC-MS in both liquid chromatography solvent with 14NH3-acetate buffer, and in solvent with the buffer modified with 15NH3-formate. Buffer switching results in characteristic mass and signal intensity changes for adduct peaks, facilitating their annotation. This relatively simple and convenient chromatography modification annotated yeast metabolomics data with similar effectiveness to growing the yeast in isotope-labeled media. Application to mouse liver data annotated both known metabolite and known adduct peaks with 95% accuracy. Overall, it identified 26% of ~ 27,000 liver LC-MS features as putative metabolites, of which ~ 2600 showed HMDB or KEGG database formula match. This workflow is well-suited to biological samples that cannot be readily isotope labeled, including plants, mammalian tissues, and tumors.
    DOI:  https://doi.org/10.1021/acs.analchem.0c00985
  3. J Biol Chem. 2020 Jul 01. pii: jbc.AC120.014189. [Epub ahead of print]
    Lafita-Navarro MC, Perez-Castro L, Zacharias LG, Barnes S, DeBerardinis RJ, Conacci-Sorrell M.
      The transcription factor aryl hydrocarbon receptor (AHR) drives the expression of genes involved in detoxification pathways in cells exposed to pollutants and other small molecules. Moreover, AHR supports transcriptional programs that promote ribosome biogenesis and protein synthesis in cells stimulated to proliferate by the oncoprotein MYC. Thus, AHR is necessary for the proliferation of MYC-overexpressing cells. To define metabolic pathways in which AHR cooperates with MYC in supporting cell growth, here we used LC-MS-based metabolomics to examine the metabolome of MYC-expressing cells uponAHR knockdown. We found that AHR knockdown reduced lactate, S-lactoyl-glutathione,N-acetyl-L-alanine, 2-hydroxyglutarate, and uridine-5-monophosphate (UMP) levels. Using our previously obtained RNA-seq data, we found that AHR mediates the expression of the UMP-generating enzymes dihydroorotate dehydrogenase (quinone) (DHODH) and uridine monophosphate synthetase (UMPS), as well as lactate dehydrogenase A (LDHA), establishing a mechanism by which AHR regulates lactate and UMP production in MYC-overexpressing cells. AHR knockdown in glioblastoma cells also reduced the expression of LDHA (and lactate), DHODH,and UMPS, but did not affect UMP levels, likely due to compensatory mechanisms in these cells. Our results indicate that AHR contributes to the regulation of metabolic pathways necessary for the proliferation of transformed cells.
    Keywords:  Myc (c-Myc); aryl hydrocarbon receptor (AhR) (AHR); cancer; gene regulation; glioblastoma; glycolysis; metabolism; metabolomics; oncogene; pyrimidine
    DOI:  https://doi.org/10.1074/jbc.AC120.014189
  4. Nat Commun. 2020 Jul 03. 11(1): 3326
    Ishak Gabra MB, Yang Y, Li H, Senapati P, Hanse EA, Lowman XH, Tran TQ, Zhang L, Doan LT, Xu X, Schones DE, Fruman DA, Kong M.
      Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.
    DOI:  https://doi.org/10.1038/s41467-020-17181-w
  5. Prog Lipid Res. 2020 Jun 24. pii: S0163-7827(20)30030-8. [Epub ahead of print]79 101050
    Schömel N, Geisslinger G, Wegner MS.
      A growing number of studies describe a connection between glycosphingolipids (GSLs) and glutamine metabolism, glucose metabolism and mitochondrial dysfunction in cancer cells. Since deregulated cell energy metabolism is one of cancer cells hallmarks, investigating this connection is an important step in the development of anti-cancer therapies. GSL species are often aberrantly regulated in human cancers. They cluster in signaling platforms in the plasma membrane and organelle membranes in so called glycosphingolipid enriched microdomains (GEMs), thereby regulating cell signaling pathways. The most important glutamine transporter for epithelial cells, alanine-serine-cysteine transporter 2 (ASCT2) locates in GEMs and is regulated by GEM composition. The accumulation of glucosylceramide and lactosylceramide in mitochondria associated ER membranes (MAMs) leads to increased oxidative phosphorylation. This increases mitochondrial reactive oxygen species (ROS) levels and influences mitochondrial dynamics. Here, we review current knowledge about deregulated GSL species in cancer, GSL influence on glutamine and glucose metabolism. In addition, the role of GSLs in MAMs, oxidative phosphorylation (OXPHOS) and mitochondrial dynamics with a special focus on mechanistic target of rapamycin (mTOR) signaling is discussed. mTOR seems to play a pivotal role in the connection between GSLs and glutamine metabolism as well as in mitochondrial signaling.
    Keywords:  Glucose metabolism; Glutamine metabolism; Mitochondria associated ER membrane; ROS; UGCG; mTOR
    DOI:  https://doi.org/10.1016/j.plipres.2020.101050
  6. Mol Cell Proteomics. 2020 07 01. pii: mcp.R120.002090. [Epub ahead of print]
    Cao W, Liu M, Kong S, Wu M, Zhang Y, Yang P.
      Intact glycopeptide identification has long been known as a key and challenging barrier to the comprehensive and accurate understanding the role of glycosylation in an organism. Intact glycopeptide analysis is a blossoming field that has received increasing attention in recent years. Mass spectrometry (MS)-based strategies and relative software tools are major drivers that have greatly facilitated the analysis of intact glycopeptides, particularly intact N-glycopeptides. This manuscript provides a systematic review of the intact glycopeptide identification process using mass spectrometry data generated in shotgun proteomic experiments, which typically focus on N-glycopeptide analysis. Particular attention is paid to the software tools that have been recently developed in the last decade for the interpretation and quality control of glycopeptide spectra acquired using different MS strategies. The review also provides information about the characteristics and applications of these software tools, discusses their advantages and disadvantages, and concludes with a discussion of outstanding tools.
    Keywords:  Glycoproteins*; Glycoproteomics; Glycosylation; Mass Spectrometry; Post-translational modifications*; computational methods; software tools
    DOI:  https://doi.org/10.1074/mcp.R120.002090
  7. Arch Toxicol. 2020 Jun 30.
    van der Stel W, Carta G, Eakins J, Darici S, Delp J, Forsby A, Bennekou SH, Gardner I, Leist M, Danen EHJ, Walker P, van de Water B, Jennings P.
      Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.
    Keywords:  ECAR; ETC; HepG2; MMP; Mitochondria; RPTEC/TERT1; Seahorse
    DOI:  https://doi.org/10.1007/s00204-020-02792-5
  8. Signal Transduct Target Ther. 2020 Jun 30. 5(1): 108
    Li D, Li Y.
      Ferroptosis is a new form of programmed cell death characterized by the accumulation of iron-dependent lethal lipid peroxides. Recent discoveries have focused on alterations that occur in lipid metabolism during ferroptosis and have provided intriguing insights into the interplay between ferroptosis and lipid metabolism in cancer. Their interaction regulates the initiation, development, metastasis, therapy resistance of cancer, as well as the tumor immunity, which offers several potential strategies for cancer treatment. This review is a brief overview of the features characterizing the interaction between ferroptosis and lipid metabolism, and highlights the significance of this interaction in cancer.
    DOI:  https://doi.org/10.1038/s41392-020-00216-5
  9. Mol Cell Proteomics. 2020 Jun 29. pii: mcp.RA120.002055. [Epub ahead of print]
    Karayel O, Tonelli F, Virreira Winter S, Geyer PE, Fan Y, Sammler EM, Alessi D, Steger M, Mann M.
      Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared to healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.
    Keywords:  Biomarker: Diagnostic; Clinical proteomics; Neurodegenerative diseases*; Parallel reaction monitoring; Parkinson's disease; Phosphorylation; Protein kinases*; Selected Ion Monitoring; Targeted mass spectrometry; Targeted therapies
    DOI:  https://doi.org/10.1074/mcp.RA120.002055
  10. Cell Syst. 2020 Jun 02. pii: S2405-4712(20)30197-6. [Epub ahead of print]
    Messner CB, Demichev V, Wendisch D, Michalick L, White M, Freiwald A, Textoris-Taube K, Vernardis SI, Egger AS, Kreidl M, Ludwig D, Kilian C, Agostini F, Zelezniak A, Thibeault C, Pfeiffer M, Hippenstiel S, Hocke A, von Kalle C, Campbell A, Hayward C, Porteous DJ, Marioni RE, Langenberg C, Lilley KS, Kuebler WM, Mülleder M, Drosten C, Suttorp N, Witzenrath M, Kurth F, Sander LE, Ralser M.
      The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.
    Keywords:  COVID-19 infection; SWATH-MS; antiviral immune response; clinical classifiers; high-throughput proteomics; mass spectrometry
    DOI:  https://doi.org/10.1016/j.cels.2020.05.012
  11. Epigenetics. 2020 Jul 02. 1-9
    Gosselt HR, Griffioen PH, van Zelst BD, Oosterom N, de Jonge R, Heil SG.
      BACKGROUND: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation.METHODS: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, -20°C and -80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models.
    RESULTS: global DNA methylation was stable over 18 months in blood at -20°C and -80°C and DNA at 4°C and -80°C. However, at 18 months DNA methylation from DNA stored at -20°C relatively decreased -6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze - thaw cycles.
    CONCLUSION: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of  small differences occuring during storage depend on the expected effect size and research question.
    Keywords:  DNA methylation; chromatography liquid; classification: DNA methylation; epigenome; humans; tandem mass spectrometry
    DOI:  https://doi.org/10.1080/15592294.2020.1786318
  12. Cancer Discov. 2020 Jun 30. pii: CD-20-0226. [Epub ahead of print]
    Nagashima H, Lee CK, Tateishi K, Higuchi F, Subramanian M, Rafferty S, Melamed L, Miller JJ, Wakimoto H, Cahill DP.
      Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor metabolite and is the currency of metabolic transactions critical for cell survival. Depending on tissue context and genotype, cancer cells have unique dependencies on NAD+ metabolic pathways. Poly(ADP-ribose) polymerases (PARPs) catalyze oligomerization of NAD+ monomers into poly(ADP-ribose) (PAR) chains during cellular response to alkylating chemotherapeutics, including procarbazine or temozolomide. Here, we find that, in endogenous IDH1 mutant tumor models, alkylator-induced cytotoxicity is markedly augmented by pharmacologic inhibition or genetic knockout of the PAR breakdown enzyme poly(ADP-ribose) glycohydrolase (PARG). Both in vitro and in vivo, we observe that concurrent alkylator and PARG inhibition depletes freely available NAD+ by preventing PAR breakdown, resulting in NAD+ sequestration and collapse of metabolic homeostasis. This effect reversed with NAD+ rescue supplementation, confirming the mechanistic basis of cytotoxicity. Thus, alkylating chemotherapy exposes a genotype-specific metabolic weakness in tumor cells that can be exploited by PARG inactivation.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0226
  13. Methods Mol Biol. 2020 ;2164 109-119
    Korach-André M.
      Fat distribution, on top of general obesity, contributes to the severity of histologic features in patients with nonalcoholic fatty liver diseases (NAFLD); and visceral obesity has been correlated to fatty liver diseases. Therefore, investigation of fat distribution in vivo could be a good predictor of fatty liver risks in obesity. Fatty acids composition is a key player in hepatic dysfunctions and cardiovascular risk in obesity. Because fatty acids can damage biological membranes, fatty acid accumulation in the liver may be partially responsible for the functional and morphological changes that are observed in NAFLD. Fatty acids stored into triglycerides are lipid species that act as signaling molecules and therefore are key regulators of posttranslational regulation of biological functions such as lipid homeostasis and lipotoxicity. Here, we describe magnetic resonance methods to investigate in vivo whole-body fat distribution and hepatic liver fatty acid composition in order to directly assess the liver metabolic status and may allow to anticipate liver diseases.
    Keywords:  Fat distribution; In vivo; Lipids; Liver steatosis; Magnetic resonance imaging; Magnetic resonance spectroscopy; Nuclear magnetic resonance; Obesity
    DOI:  https://doi.org/10.1007/978-1-0716-0704-6_12
  14. Sci Rep. 2020 Jul 02. 10(1): 10918
    Lin Y, Caldwell GW, Li Y, Lang W, Masucci J.
      There is a long-standing concern for the lack of reproducibility of the untargeted metabolomic approaches used in pharmaceutical research. Two types of human plasma samples were split into two batches and analyzed in two individual labs for untargeted GC-MS metabolomic profiling. The two labs used the same silylation sample preparation protocols but different instrumentation, data processing software, and database. There were 55 metabolites annotated reproducibly, independent of the labs. The median coefficient variations (CV%) of absolute spectra ion intensities in both labs were less than 30%. However, the comparison of normalized ion intensity among biological groups, were inconsistent across labs. Predicted power based on annotated metabolites was evaluated post various normalization, data transformation and scaling. For the first time our study reveals the numerical details about the variations in metabolomic annotation and relative quantification using plain inter-laboratory GC-MS untargeted metabolomic approaches. Especially we compare several commonly used post-acquisition strategies and found normalization could not strengthen the annotation accuracy or relative quantification precision of untargeted approach, instead it will impact future experimental design. Standardization of untargeted metabolomics protocols, including sample preparation, instrumentation, data processing, etc., is critical for comparison of untargeted data across labs.
    DOI:  https://doi.org/10.1038/s41598-020-67939-x
  15. Trends Cancer. 2020 Jul;pii: S2405-8033(20)30084-4. [Epub ahead of print]6(7): 593-604
    Attané C, Muller C.
      Over the past decade, it has become apparent that metabolic reprogramming is a key event in tumor progression. The tumor microenvironment (TME) is a source of metabolites for tumor cells. Lipid-filled mature adipocytes are frequently found in proximity to invasive human tumors and release free fatty acids (FFAs) through lipolysis. These FFAs are taken up by tumor cells and used to promote tumor progression by mechanisms that include mitochondrial fatty acid oxidation (FAO). This review discusses recent advances in our understanding of this metabolic symbiosis between adipocytes and cancer cells and underlines the differences in this metabolic crosstalk between the various types of cancer and their localization.
    Keywords:  adipocyte; cancer metabolism; fatty acids; metabolic crosstalk
    DOI:  https://doi.org/10.1016/j.trecan.2020.03.001
  16. Anal Chem. 2020 Jun 30.
    Kuhring M, Eisenberger A, Schmidt V, Kränkel N, Leistner DM, Kirwan J, Beule D.
      Targeted quantitative mass spectrometry metabolite profiling is the workhorse of metabolomics research. Robust and reproducible data is essential for confidence in analytical results and is particularly important with large-scale studies. Commercial kits are now available which use carefully calibrated and validated internal and external standards to provide such reliability. However, they are still subject to processing and technical errors in their use and should be subject to a laboratory's routine quality assurance and quality control measures to maintain confidence in the results. We discuss important systematic and random measurement errors when using these kits and suggest measures to detect and quantify them. We demonstrate how wider analysis of the entire data set alongside standard analyses of quality control samples can be used to identify outliers and quantify systematic trends to improve downstream analysis. Finally, we present the MeTaQuaC software which implements the above concepts and methods for Biocrates kits and other target data sets and creates a comprehensive quality control report containing rich visualization and informative scores and summary statistics. Preliminary unsupervised multivariate analysis methods are also included to provide rapid insight into study variables and groups. MeTaQuaC is provided as an open source R package under a permissive MIT license and includes detailed user documentation.
    DOI:  https://doi.org/10.1021/acs.analchem.0c00136
  17. Front Cell Dev Biol. 2020 ;8 385
    Abooshahab R, Hooshmand K, Razavi SA, Gholami M, Sanoie M, Hedayati M.
      One of the challenges in the area of diagnostics of human thyroid cancer is a preoperative diagnosis of thyroid nodules with indeterminate cytology. Herein, we report an untargeted metabolomics analysis to identify circulating thyroid nodule metabolic signatures, to find new novel metabolic biomarkers. Untargeted gas chromatography-quadrupole-mass spectrometry was used to ascertain the specific plasma metabolic changes of thyroid nodule patients, which consisted of papillary thyroid carcinoma (PTC; n = 19), and multinodular goiter (MNG; n = 16), as compared to healthy subjects (n = 20). Diagnostic models were constructed using multivariate analyses such as principal component analysis, orthogonal partial least squares-discriminant analysis, and univariate analysis including One-way ANOVA and volcano plot by MetaboAnalyst and SIMCA software. Because of the multiple-testing issue, false discovery rate p-values were also computed for these functions. A total of 60 structurally annotated metabolites were subjected to statistical analysis. A combination of univariate and multivariate statistical analyses revealed a panel of metabolites responsible for the discrimination between thyroid nodules and healthy subjects, with variable importance in the projection (VIP) value greater than 0.8 and p-value less than 0.05. Significantly altered metabolites between thyroid nodules versus healthy persons are those associated with amino acids metabolism, the tricarboxylic acid cycle, fatty acids, and purine and pyrimidine metabolism, including cysteine, cystine, glutamic acid, α-ketoglutarate, 3-hydroxybutyric acid, adenosine-5-monophosphate, and uracil, respectively. Further, sucrose metabolism differed profoundly between thyroid nodule patients and healthy subjects. Moreover, according to the receiver operating characteristic (ROC) curve analysis, sucrose could discriminate PTC from MNG (area under ROC curve value = 0.92). This study enhanced our understanding of the distinct metabolic pathways associated with thyroid nodules, which enabled us to distinguish between patients and healthy subjects. In addition, our study showed extensive sucrose metabolism in the plasma of thyroid nodule patients, which provides a new metabolic signature of the thyroid nodule's tumorigenesis. Accordingly, it suggests that sucrose can be considered as a circulating biomarker for differential diagnosis between malignancy and benignity in indeterminate thyroid nodules.
    Keywords:  GC-MS; metabolomics; multinodular goiter; papillary thyroid cancer; thyroid nodules
    DOI:  https://doi.org/10.3389/fcell.2020.00385
  18. Methods Mol Biol. 2020 ;2156 187-202
    Song Y, Vu HS, Shiva S, Fruehan C, Roth MR, Tamura P, Welti R.
      Lipid changes that occur in leaves of plants (e.g., Arabidopsis thaliana), during cold and freezing stress can be analyzed with electrospray ionization triple quadrupole mass spectrometry, using high-throughput multiple reaction monitoring (MRM). An online tool, LipidomeDB Data Calculation Environment, is employed for mass spectral data processing.
    Keywords:  Cold; Freezing; Lipidomics; Lipidomics data processing; Mass spectrometry; Multiple reaction monitoring (MRM); Postfreezing recovery
    DOI:  https://doi.org/10.1007/978-1-0716-0660-5_14
  19. Mol Cell Proteomics. 2020 Jul 02. pii: mcp.TIR120.002048. [Epub ahead of print]
    Yu F, Haynes SE, Teo GC, Avtonomov DM, Polasky DA, Nesvizhskii AI.
      Ion mobility brings an additional dimension of separation to liquid chromatography-mass spectrometry, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to time-of-flight mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most commonly used to analyze these data. However, due to the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4-D feature extraction and quantification. Using a HeLa data set published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (~30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (~10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification precision and accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two-hour PASEF run in under 70 minutes on a typical desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-enzymatic searching, we significantly increase the number of identified peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation prior to MS/MS analysis.
    Keywords:  Algorithms; Bioinformatics; Ion Mobility; Label-free quantification; PASEF; Protein Identification*; Tandem Mass Spectrometry
    DOI:  https://doi.org/10.1074/mcp.TIR120.002048
  20. Metabolites. 2020 Jun 28. pii: E268. [Epub ahead of print]10(7):
    Achkar IW, Kader S, Dib SS, Junejo K, Al-Bader SB, Hayat S, Bhagwat AM, Rousset X, Wang Y, Viallet J, Suhre K, Halama A.
      BACKGROUND: Dysregulated cancer metabolism is associated with acquired resistance to chemotherapeutic treatment and contributes to the activation of cancer survival mechanisms. However, which metabolic pathways are activated following treatment often remains elusive. The combination of chicken embryo tumor models (in ovo) with metabolomics phenotyping could offer a robust platform for drug testing. Here, we assess the potential of this approach in the treatment of an in ovo triple negative breast cancer with doxorubicin.METHODS: MB-MDA-231 cells were grafted in ovo. The resulting tumors were then treated with doxorubicin or dimethyl sulfoxide (DMSO) for six days. Tumors were collected and analyzed using a global untargeted metabolomics and comprehensive lipidomics.
    RESULTS: We observed a significant suppression of tumor growth in the doxorubicin treated group. The metabolic profiles of doxorubicin and DMSO-treated tumors were clearly separated in a principle component analysis. Inhibition of glycolysis, nucleotide synthesis, and glycerophospholipid metabolism appear to be triggered by doxorubicin treatment, which could explain the observed suppressed tumor growth. In addition, metabolic cancer survival mechanisms could be supported by an acceleration of antioxidative pathways.
    CONCLUSIONS: Metabolomics in combination with in ovo tumor models provide a robust platform for drug testing to reveal tumor specific treatment targets such as the antioxidative tumor capacity.
    Keywords:  cancer survival mechanism; chicken chorioallantoic membrane (CAM) system in ovo model; doxorubicin treatment; lipidomics; metabolomics; triple negative breast cancer
    DOI:  https://doi.org/10.3390/metabo10070268
  21. Clin Nutr. 2020 Jun 04. pii: S0261-5614(20)30277-6. [Epub ahead of print]
    Bellissimo MP, Ziegler TR, Jones DP, Liu KH, Fernandes J, Roberts JL, Weitzmann MN, Pacifici R, Alvarez JA.
      BACKGROUND & AIMS: There is a considerable degree of variation in bone mineral density (BMD) within populations. Use of plasma metabolomics may provide insight into established and novel determinants of BMD variance, such as nutrition and gut microbiome composition, to inform future prevention and treatment strategies for loss of BMD. Using high-resolution metabolomics (HRM), we examined low-molecular weight plasma metabolites and nutrition-related metabolic pathways associated with BMD.METHODS: This cross-sectional study included 179 adults (mean age 49.5 ± 10.3 yr, 64% female). Fasting plasma was analyzed using ultra-high-resolution mass spectrometry with liquid chromatography. Whole body and spine BMD were assessed by dual energy X-ray absorptiometry and expressed as BMD (g/cm2) or Z-scores. Multiple linear regression, pathway enrichment, and module analyses were used to determine key plasma metabolic features associated with bone density.
    RESULTS: Of 10,210 total detected metabolic features, whole body BMD Z-score was associated with 710 metabolites, which were significantly enriched in seven metabolic pathways, including linoleic acid, fatty acid activation and biosynthesis, and glycerophospholipid metabolism. Spine BMD was associated with 970 metabolites, significantly enriched in pro-inflammatory pathways involved in prostaglandin formation and linoleic acid metabolism. In module analyses, tryptophan- and polyamine-derived metabolites formed a network that was significantly associated with spine BMD, supporting a link with the gut microbiome.
    CONCLUSIONS: Plasma HRM provides comprehensive information relevant to nutrition and components of the microbiome that influence bone health. This data supports pro-inflammatory fatty acids and the gut microbiome as novel regulators of postnatal bone remodeling.
    Keywords:  Fats; Inflammation; Nutrition; Omega-6; PUFA; Skeletal health
    DOI:  https://doi.org/10.1016/j.clnu.2020.05.041
  22. J Proteome Res. 2020 Jul 03.
    Wang Q, Su B, Dong L, Jiang T, Tan Y, Lu X, Liu X, Lin X, Xu G.
      Assessment and prediction of prognostic risk in patients with hepatocellular carcinoma (HCC) would greatly benefit the optimal treatment selection. Here, we aimed to identify the critical metabolites associated with the outcomes and develop a risk score to assess the prognosis of HCC patients after curative resection. A total of 78 serum samples of HCC patients were analyzed by liquid chromatography-mass spectrometry (LC-MS) to characterize the metabolic profiling. A novel network-based feature selection method (NFSM) was developed to define the critical metabolites with the most discriminant capacity to outcomes. The metabolites defined by NFSM was further reduced by Cox regression analysis to generate a prognostic metabolite panel-phenylalanine and choline. Furthermore, univariate and multivariate Cox regression analyses were applied to combine the metabolite panel with the presence of satellite nodes to generate a global prognostic index (GPI) score for overall survival assessment. Compared with the current clinical classification systems including Barcelona-Clinic Liver Cancer (BCLC) stage, Tumor-Node-Metastasis (TNM) stage and Albumin-Bilirubin (ALBI) grade, the GPI score presented comparable performance according to the time-dependent receiver operating characteristics curves and was validated in an independent cohort, which suggested metabolomics could serve as a helpful tool to stratify the HCC prognostic risk after operation.
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00344
  23. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jun 13. pii: S1570-0232(20)30185-9. [Epub ahead of print]1152 122243
    du Toit T, van Rooyen D, Stander MA, Atkin SL, Swart AC.
      The C11-oxy androgens have been implicated in the progression of many diseases and endocrine-linked disorders, such as polycystic ovarian syndrome (PCOS), congenital adrenal hyperplasia, specifically 21-hydroxylase deficiency (21OHD), castration resistant prostate cancer (CRPC), as well as premature adrenarche. While the C11-oxy C19 steroids have been firmly established in the steroid arena, the C11-oxy C21 steroids are now also of significance. The current study reports on a high-throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) method for the separation and quantification of 52 steroids in peripheral serum, which include the C11-oxy C19 and C11-oxy C21 steroids. Fifteen deuterium-labelled steroids were included for absolute quantification, which incorporates steroid extraction efficiency, together with one steroid and four non-steroidal compounds serving as quality controls (QC). The 15 min run-time per sample (16 min injection-to-injection time with an 8-step gradient) quantifies 68 analytes in a 2 µL injection volume. A single chromatographic step simultaneously identifies steroids in the mineralocorticoid, glucocorticoid and androgen pathways in adrenal steroidogenesis, together with steroid metabolites produced in the periphery, presenting an analytical method for the application of screening in vivo clinical samples. This study highlights cross-talk between the C11-oxy steroids, and describes the optimisation of multiple reaction monitoring required to measure steroids accurately. The limit of detection for the steroid metabolites ranged from 0.002 to 20 ng/mL and the limit of quantification from 0.02 to 100 ng/mL. The calibration range for the steroids ranged from 0.002 to 1000 ng/mL and for the QC compounds from 0.075 to 750 ng/mL. The method is fully validated in terms of accuracy (%RSD, <13%), precision (including inter-day variability across a three-day period) (%RSD, <16%), recovery (average 102.42%), matrix effect (ranging from -15.25 to 14.25%) and process efficiency (average 101.79%). The dilution protocol for the steroids, internal standards and QC compounds were validated, while the ion ratios of the steroid metabolites (%RSD, <16%) and QC compounds were monitored and the accuracy bias values (%RSD, <9%) were within acceptable limits. The method was subsequently used to quantify steroid levels in a cohort of healthy women. C11-oxy steroid metabolites produced as intermediates in steroidogenic pathways, together with end-products included in the method can potentially characterise the 11β-hydroxyandrostenedione-, C21- and C11-oxy backdoor pathways in vivo. The identification of these C11-oxy C19 and C11-oxy C21 intermediates would allow insight into active pathways, while steroid metabolism could be traced in patients and reference ranges established in both normal and abnormal conditions. Furthermore, conditions currently undefined in terms of the C11-oxy steroids would benefit from the analysis provided by this method, while the C11-oxy steroids could be further explored in PCOS, 21OHD, CRPC and adrenarche.
    Keywords:  11keto-testosterone (11KT); 11β-hydroxyandrostenedione (11OHA4); Castration resistant prostate cancer (CRPC); Congenital adrenal hyperplasia (CAH); Oxygenated; Polycystic ovary syndrome (PCOS)
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122243
  24. J Biomed Res. 2019 Oct 31. 1-11
    Yi H, Talmon G, Wang J.
      Glutamine and glutamate are major bioenergy substrates for normal and cancer cell growth. Cancer cells need more biofuel than normal tissues for energy supply, anti-oxidation activity and biomass production. Genes related to metabolic chains in many cancers are somehow mutated, which makes cancer cells more glutamate dependent. Meanwhile, glutamate is an excitatory neurotransmitter for conducting signals through binding with different types of receptors in central neuron system. Interestingly, increasing evidences have shown involvement of glutamate signaling, guided through their receptors, in human malignancy. Dysregulation of glutamate transporters, such as excitatory amino acid transporter and cystine/glutamate antiporter system, also generates excessive extracellular glutamate, which in turn, activates glutamate receptors on cancer cells and results in malignant growth. These features make glutamate an attractive target for anti-cancer drug development with some glutamate targeted but blood brain barrier impermeable anti-psychosis drugs under consideration. We discussed the relevant progressions and drawbacks in this field herein.
    Keywords:  cancers; glutamate; glutamate receptors; glutamate transporters; metabolism
    DOI:  https://doi.org/10.7555/JBR.34.20190037
  25. Biology (Basel). 2020 Jun 26. pii: E140. [Epub ahead of print]9(6):
    Stransky S, Aguilan J, Lachowicz J, Madrid-Aliste C, Nieves E, Sidoli S.
      Chromatin accessibility is a major regulator of gene expression. Histone writers/erasers have a critical role in chromatin compaction, as they "flag" chromatin regions by catalyzing/removing covalent post-translational modifications on histone proteins. Anomalous chromatin decondensation is a common phenomenon in cells experiencing aging and viral infection. Moreover, about 50% of cancers have mutations in enzymes regulating chromatin state. Numerous genomics methods have evolved to characterize chromatin state, but the analysis of (in)accessible chromatin from the protein perspective is not yet in the spotlight. We present an overview of the most used approaches to generate data on chromatin accessibility and then focus on emerging methods that utilize mass spectrometry to quantify the accessibility of histones and the rest of the chromatin bound proteome. Mass spectrometry is currently the method of choice to quantify entire proteomes in an unbiased large-scale manner; accessibility on chromatin of proteins and protein modifications adds an extra quantitative layer to proteomics dataset that assist more informed data-driven hypotheses in chromatin biology. We speculate that this emerging new set of methods will enhance predictive strength on which proteins and histone modifications are critical in gene regulation, and which proteins occupy different chromatin states in health and disease.
    Keywords:  DNA methylation; chromatin; histone; mass spectrometry; post-translational modification; proteome
    DOI:  https://doi.org/10.3390/biology9060140
  26. Essays Biochem. 2020 Jul 03. pii: EBC20190082. [Epub ahead of print]
    Christie WW, Harwood JL.
      The chemistry, biochemistry, pharmacology and molecular biology of oxylipins (defined as a family of oxygenated natural products that are formed from unsaturated fatty acids by pathways involving at least one step of dioxygen-dependent oxidation) are complex and occasionally contradictory subjects that continue to develop at an extraordinarily rapid rate. The term includes docosanoids (e.g. protectins, resolvins and maresins, or specialized pro-resolving mediators), eicosanoids and octadecanoids and plant oxylipins, which are derived from either the omega-6 (n-6) or the omega-3 (n-3) families of polyunsaturated fatty acids. For example, the term eicosanoid is used to embrace those biologically active lipid mediators that are derived from C20 fatty acids, and include prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acids and related oxygenated derivatives. The key enzymes for the production of prostanoids are prostaglandin endoperoxide H synthases (cyclo-oxygenases), while lipoxygenases and oxidases of the cytochrome P450 family produce numerous other metabolites. In plants, the lipoxygenase pathway from C18 polyunsaturated fatty acids yields a variety of important products, especially the jasmonates, which have some comparable structural features and functions. Related oxylipins are produced by non-enzymic means (isoprostanes), while fatty acid esters of hydroxy fatty acids (FAHFA) are now being considered together with the oxylipins from a functional perspective. In all kingdoms of life, oxylipins usually act as lipid mediators through specific receptors, have short half-lives and have functions in innumerable biological contexts.
    Keywords:  fatty acid metabolism; lipid mediators; polyunsaturated fatty acids
    DOI:  https://doi.org/10.1042/EBC20190082