bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒11‒29
28 papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh
  1. Quantitative Proteomics in Drosophila with Holidic Stable-Isotope Labeling of Amino Acids in Fruit Flies (SILAF).
  2. Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry.
  3. The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation.
  4. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis.
  5. SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.
  6. Transcriptomic analysis suggested the involvement of impaired lipid droplet biogenesis in graphene oxide-induced cytotoxicity in human umbilical vein endothelial cells.
  7. Comparable Plasma Lipid Changes in Patients with High-Grade Cervical Intraepithelial Neoplasia and Patients with Cervical Cancer.
  8. Potential impact of tissue molecular heterogeneity on ambient mass spectrometry profiles: a note of caution in choosing the right disease model.
  9. Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Clinical Metabolic Phenotyping Assays.
  10. Evaluating and minimizing batch effects in metabolomics.
  11. Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra.
  12. Top-down Proteomics: Challenges, Innovations, and Applications in Basic and Clinical Research.
  13. Convergence of Plasma Metabolomics and Proteomics Analysis to Discover Signatures of High-Grade Serous Ovarian Cancer.
  14. Supercritical fluid chromatography - Mass spectrometry in metabolomics: Past, present, and future perspectives.
  15. Nuclear receptors and transcriptional regulation in non-alcoholic fatty liver disease.
  16. The Power of Plasticity-Metabolic Regulation of Hepatic Stellate Cells.
  17. Quantitative shotgun proteome analysis by direct infusion.
  18. The influence of secreted factors and extracellular vesicles in ovarian cancer metastasis.
  19. Mouse Age Matters: How Age Affects the Murine Plasma Metabolome.
  20. Metabolic Compartmentalization at the Leading Edge of Metastatic Cancer Cells.
  21. Profiling of Low-Molecular-Weight Carbonyls and Protein Modifications in Flavored Milk.
  22. Multi-omics Analysis of CRISPRi-Knockdowns Identifies Mechanisms that Buffer Decreases of Enzymes in E. coli Metabolism.
  23. Metabolomics meets lipidomics: Assessing the small molecule component of metabolism.
  24. Dietary Energy Modulation and Autophagy: Exploiting Metabolic Vulnerabilities to Starve Cancer.
  25. Characterization of Phosphorylated Proteins Using Mass Spectrometry.
  26. DGAT1 protects tumor from lipotoxicity, emerging as a promising metabolic target for cancer therapy.
  27. Comprehensive lipid analysis of green Arabica coffee beans by LC-HRMS/MS.
  28. A simple LC-MS/MS method for the simultaneous quantification of endocannabinoids in biological samples.
  1. Methods Mol Biol. 2021 ;2192 75-87
    Quantitative Proteomics in Drosophila with Holidic Stable-Isotope Labeling of Amino Acids in Fruit Flies (SILAF).
    Florian A Schober, Ilian Atanassov, Christoph Freyer, Anna Wredenberg.
      Protein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.
    Keywords:  Drosophila; Proteomics; SILAC, Mitochondria; Stable-isotope labeling
    DOI:  https://doi.org/10.1007/978-1-0716-0834-0_7
  2. Nat Commun. 2020 Nov 27. 11(1): 6035
    Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry.
    Danye Qiu, Miranda S Wilson, Verena B Eisenbeis, Robert K Harmel, Esther Riemer, Thomas M Haas, Christopher Wittwer, Nikolaus Jork, Chunfang Gu, Stephen B Shears, Gabriel Schaaf, Bernd Kammerer, Dorothea Fiedler, Adolfo Saiardi, Henning J Jessen.
      The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.
    DOI:  https://doi.org/10.1038/s41467-020-19928-x
  3. Exp Mol Med. 2020 Nov 25.
    The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation.
    Inhyub Kim, Minkyoung Kim, Min Kyung Park, Ravi Naik, Jae Hyung Park, Bo-Kyung Kim, Yongseok Choi, Kwan Young Chang, Misun Won, Hyun Seung Ban, Kyeong Lee.
      Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.
    DOI:  https://doi.org/10.1038/s12276-020-00523-5
  4. Proc Natl Acad Sci U S A. 2020 Nov 23. pii: 202017152. [Epub ahead of print]
    Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis.
    Junmei Yi, Jiajun Zhu, Jiao Wu, Craig B Thompson, Xuejun Jiang.
      Ferroptosis, a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various signaling pathways related to cancer. In this study, we found that activating mutation of phosphatidylinositol 3-kinase (PI3K) or loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function, highly frequent events in human cancer, confers ferroptosis resistance in cancer cells, and that inhibition of the PI3K-AKT-mTOR signaling axis sensitizes cancer cells to ferroptosis induction. Mechanistically, this resistance requires sustained activation of mTORC1 and the mechanistic target of rapamycin (mTOR)C1-dependent induction of sterol regulatory element-binding protein 1 (SREBP1), a central transcription factor regulating lipid metabolism. Furthermore, stearoyl-CoA desaturase-1 (SCD1), a transcriptional target of SREBP1, mediates the ferroptosis-suppressing activity of SREBP1 by producing monounsaturated fatty acids. Genetic or pharmacologic ablation of SREBP1 or SCD1 sensitized ferroptosis in cancer cells with PI3K-AKT-mTOR pathway mutation. Conversely, ectopic expression of SREPB1 or SCD1 restored ferroptosis resistance in these cells, even when mTORC1 was inhibited. In xenograft mouse models for PI3K-mutated breast cancer and PTEN-defective prostate cancer, the combination of mTORC1 inhibition with ferroptosis induction resulted in near-complete tumor regression. In conclusion, hyperactive mutation of PI3K-AKT-mTOR signaling protects cancer cells from oxidative stress and ferroptotic death through SREBP1/SCD1-mediated lipogenesis, and combination of mTORC1 inhibition with ferroptosis induction shows therapeutic promise in preclinical models.
    Keywords:  SREBP1; cancer; ferroptosis; lipogenesis; mTOR
    DOI:  https://doi.org/10.1073/pnas.2017152117
  5. Nat Commun. 2020 11 23. 11(1): 5927
    SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease.
    Philipp Gut, Sanna Matilainen, Jesse G Meyer, Pieti Pällijeff, Joy Richard, Christopher J Carroll, Liliya Euro, Christopher B Jackson, Pirjo Isohanni, Berge A Minassian, Reem A Alkhater, Elsebet Østergaard, Gabriele Civiletto, Alice Parisi, Jonathan Thevenet, Matthew J Rardin, Wenjuan He, Yuya Nishida, John C Newman, Xiaojing Liu, Stefan Christen, Sofia Moco, Jason W Locasale, Birgit Schilling, Anu Suomalainen, Eric Verdin.
      Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-β (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
    DOI:  https://doi.org/10.1038/s41467-020-19743-4
  6. Chem Biol Interact. 2020 Nov 19. pii: S0009-2797(20)31828-7. [Epub ahead of print] 109325
    Transcriptomic analysis suggested the involvement of impaired lipid droplet biogenesis in graphene oxide-induced cytotoxicity in human umbilical vein endothelial cells.
    Yingmei Luo, Xuefeng Wang, Yi Cao.
      Previous studies revealed that direct contact with graphene oxide (GO) induced cytotoxic effects, but the importance of involvement of metabolic pathways, in particular lipid metabolism pathways, might be overlooked. In this study, human umbilical vein endothelial cells (HUVECs) were exposed to GO with large size (denoted as GO-L) or small size (denoted as GO-S), and transcriptomics were used to understand the mechanisms of cytotoxicity of GO at systemic levels. It was shown that GO-L more significantly induced cytotoxicity compared with GO-S. Transcriptomic analysis revealed that compared with GO-S, GO-L had larger impact on gene ontology terms related with mitochondrial function as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related with cell death and growth. But GO-S showed greater influence on KEGG pathways related with lipid metabolism. Both types of GO showed minimal impact on oxidative stress but increased de novo lipogenesis protein fatty acid synthase (FASN). However, only GO-S significantly promoted acyl-CoA synthetase 3 (ACSL3), a key enzyme responsible for esterification of free fatty acids and lipid droplet biogenesis. Not surprisingly, GO-L but not GO-S impaired lipid droplet biogenesis, and increasing lipid levels by oleic acid or α-linolenic acid reduced the cytotoxicity of GO-L to HUVECs. Combined, the results from this study suggested that impaired lipid droplet biogenesis was involved in GO-induced cytotoxicity in HUVECs, and inducing lipid droplet biogenesis could prevent the cytotoxicity of GO.
    Keywords:  Acyl-CoA synthetase (ACSL); Graphene oxide (GO); Human umbilical vein endothelial cells (HUVECs); Lipid droplets; Transcriptomics
    DOI:  https://doi.org/10.1016/j.cbi.2020.109325
  7. J Proteome Res. 2020 Nov 26.
    Comparable Plasma Lipid Changes in Patients with High-Grade Cervical Intraepithelial Neoplasia and Patients with Cervical Cancer.
    Miso Nam, Sang-Soo Seo, Sunhee Jung, Seo Young Jang, Jueun Lee, Minji Kwon, Imran Khan, Do Hyun Ryu, Mi Kyung Kim, Geum-Sook Hwang.
      Cervical cancer is the fourth most prevalent cancer among women worldwide and usually develops from cervical intraepithelial neoplasia (CIN). In the present study, we compared alterations in lipids associated with high-grade CIN and cervical cancer with those associated with a normal status and low-grade CIN by performing global lipid profiling on plasma (66 healthy controls and 55 patients with CIN1, 44 with CIN2/3, and 60 with cervical cancer) using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry. We identified 246 lipids and found 31 lipids with similar alterations in both high-grade CIN and cervical cancer. Among these 31 lipids, four lipid classes (phosphatidylcholine, phosphatidylethanolamine, diglyceride, and free fatty acids) were identified as the major lipid classes with significant differences in the patients with CIN2/3 and cervical cancer compared to the healthy controls and the patients with CIN1. Lipid metabolites belonging to the same classes were positively correlated with each other. High-grade CIN and cervical cancer induce comparable changes in lipid levels, which are closely related to the development of cervical tumors. These results suggest that lipid profiling is a useful method for monitoring progression to cervical cancer.
    Keywords:  cervical cancer; cervical intraepithelial neoplasia; global lipid profiling; lipid metabolism; liquid chromatography−mass spectrometry
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00640
  8. Anal Bioanal Chem. 2020 Nov 27.
    Potential impact of tissue molecular heterogeneity on ambient mass spectrometry profiles: a note of caution in choosing the right disease model.
    Lauren Katz, Michael Woolman, Alessandra Tata, Arash Zarrine-Afsar.
      This review provides a summary of known molecular alterations in commonly used cancer models and strives to stipulate how they may affect ambient mass spectrometry profiles. Immortalized cell lines are known to accumulate mutations, and xenografts derived from cell lines are known to contain tumour microenvironment elements from the host animal. While the use of human specimens for mass spectrometry profiling studies is highly encouraged, patient-derived xenografts with low passage numbers could provide an alternative means of amplifying material for ambient MS research when needed. Similarly, genetic preservation of patient tissue seen in some organoid models, further verified by qualitative proteomic and transcriptomic analyses, may argue in favor of organoid suitability for certain ambient profiling studies. However, to choose the appropriate model, pre-evaluation of the model's molecular characteristics in the context of the research question(s) being asked will likely provide the most appropriate strategy to move research forward. This can be achieved by performing comparative ambient MS analysis of the disease model of choice against a small amount of patient tissue to verify concordance. Disease models, however, will continue to be useful tools to orthogonally validate metabolic states of patient tissues through controlled genetic alterations that are not possible with patient specimens.
    Keywords:  Ambient mass spectrometry; Animal models of cancer; Cell line models of disease; Lipid imaging and analysis; Mass spectrometry imaging; Metabolomics
    DOI:  https://doi.org/10.1007/s00216-020-03054-0
  9. J Proteome Res. 2020 Nov 25.
    Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Clinical Metabolic Phenotyping Assays.
    Andrew D Southam, Harriet Pursell, Gianfranco Frigerio, Andris Jankevics, Ralf J M Weber, Warwick B Dunn.
      Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and nonpolar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water, and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water), and a monophasic method for nonpolar compound extraction (isopropanol/water). All methods were applied to mammalian heart, kidney, and liver tissues. Polar extracts were analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) and nonpolar extracts by C18 reversed-phase UHPLC-MS. Method reproducibility and yield were assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median relative standard deviation (RSD) < 18%; negative ion: median RSD < 28%) and nonpolar (positive and negative ion: median RSD < 15%) extractions for heart, kidneys, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC-MS. The nonpolar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine, and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and are therefore most suited for future automation.
    Keywords:  metabolic profiling; metabolite profiling; tissue extraction; tissue preparation
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00660
  10. Mass Spectrom Rev. 2020 Nov 25.
    Evaluating and minimizing batch effects in metabolomics.
    Wei Han, Liang Li.
      Determining metabolomic differences among samples of different phenotypes is a critical component of metabolomics research. With the rapid advances in analytical tools such as ultrahigh-resolution chromatography and mass spectrometry, an increasing number of metabolites can now be profiled with high quantification accuracy. The increased detectability and accuracy raise the level of stringiness required to reduce or control any experimental artifacts that can interfere with the measurement of phenotype-related metabolome changes. One of the artifacts is the batch effect that can be caused by multiple sources. In this review, we discuss the origins of batch effects, approaches to detect interbatch variations, and methods to correct unwanted data variability due to batch effects. We recognize that minimizing batch effects is currently an active research area, yet a very challenging task from both experimental and data processing perspectives. Thus, we try to be critical in describing the performance of a reported method with the hope of stimulating further studies for improving existing methods or developing new methods.
    Keywords:  NMR; batch effect; mass spectrometry; metabolome analysis; metabolomics
    DOI:  https://doi.org/10.1002/mas.21672
  11. Nat Biotechnol. 2020 Nov 23.
    Systematic classification of unknown metabolites using high-resolution fragmentation mass spectra.
    Kai Dührkop, Louis-Félix Nothias, Markus Fleischauer, Raphael Reher, Marcus Ludwig, Martin A Hoffmann, Daniel Petras, William H Gerwick, Juho Rousu, Pieter C Dorrestein, Sebastian Böcker.
      Metabolomics using nontargeted tandem mass spectrometry can detect thousands of molecules in a biological sample. However, structural molecule annotation is limited to structures present in libraries or databases, restricting analysis and interpretation of experimental data. Here we describe CANOPUS (class assignment and ontology prediction using mass spectrometry), a computational tool for systematic compound class annotation. CANOPUS uses a deep neural network to predict 2,497 compound classes from fragmentation spectra, including all biologically relevant classes. CANOPUS explicitly targets compounds for which neither spectral nor structural reference data are available and predicts classes lacking tandem mass spectrometry training data. In evaluation using reference data, CANOPUS reached very high prediction performance (average accuracy of 99.7% in cross-validation) and outperformed four baseline methods. We demonstrate the broad utility of CANOPUS by investigating the effect of microbial colonization in the mouse digestive system, through analysis of the chemodiversity of different Euphorbia plants and regarding the discovery of a marine natural product, revealing biological insights at the compound class level.
    DOI:  https://doi.org/10.1038/s41587-020-0740-8
  12. Expert Rev Proteomics. 2020 Nov 24.
    Top-down Proteomics: Challenges, Innovations, and Applications in Basic and Clinical Research.
    Kyle A Brown, Jake A Melby, David S Roberts, Ying Ge.
      Introduction- A better understanding of the underlying molecular mechanism of diseases is critical for developing more effective diagnostic tools and therapeutics towards precision medicine. However, many challenges remain to unravel the complex nature of diseases. Areas covered- Changes in protein isoform expression and post-translation modifications (PTMs) have gained recognition for their role in underlying disease mechanisms. Top-down mass spectrometry (MS)-based proteomics is increasingly recognized as an important method for the comprehensive characterization of proteoforms that arise from alternative splicing events and/or PTMs for basic and clinical research. Here, we review the challenges, technological innovations, and recent studies that utilize top-down proteomics to elucidate changes in the proteome with an emphasis on its use to study the diseases of the heart. Expert Opinion- Proteoform-resolved information can substantially contribute to the understanding of the molecular mechanisms underlying various diseases and for the identification of novel proteoform targets for better therapeutic development toward precision medicine. Despite the challenges of sequencing intact proteins, top-down proteomics has enabled a wealth of information regarding protein isoform switching and changes in PTMs. Continuous developments in sample preparation, intact protein separation, and instrumentation for top-down MS have broadened its capabilities to characterize proteoforms from a range of samples on an increasingly global scale.
    Keywords:  Heart Diseases; Mass Spectrometry; Post-translational Modifications; Proteoforms; Top-down Proteomics
    DOI:  https://doi.org/10.1080/14789450.2020.1855982
  13. Cancers (Basel). 2020 Nov 19. pii: E3447. [Epub ahead of print]12(11):
    Convergence of Plasma Metabolomics and Proteomics Analysis to Discover Signatures of High-Grade Serous Ovarian Cancer.
    Hee-Sung Ahn, Jeonghun Yeom, Jiyoung Yu, Young-Il Kwon, Jae-Hoon Kim, Kyunggon Kim.
      The 5-year survival rate in the early and late stages of ovarian cancer differs by 63%. In addition, a liquid biopsy is necessary because there are no symptoms in the early stage and tissue collection is difficult without using invasive methods. Therefore, there is a need for biomarkers to achieve this goal. In this study, we found blood-based metabolite or protein biomarker candidates for the diagnosis of ovarian cancer in the 20 clinical samples (10 ovarian cancer patients and 10 healthy control subjects). Plasma metabolites and proteins were measured and quantified using mass spectrometry in ovarian cancer patients and control groups. We identified the differential abundant biomolecules (34 metabolites and 197 proteins) and statistically integrated molecules of different dimensions to better understand ovarian cancer signal transduction and to identify novel biological mechanisms. In addition, the biomarker reliability was verified through comparison with existing research results. Integrated analysis of metabolome and proteome identified emerging properties difficult to grasp with the single omics approach, more reliably interpreted the cancer signaling pathway, and explored new drug targets. Especially, through this analysis, proteins (PPCS, PMP2, and TUBB) and metabolites (L-carnitine and PC-O (30:0)) related to the carnitine system involved in cancer plasticity were identified.
    Keywords:  FIA–MS/MS; LC–MS/MS; OMICS integrated analysis; biomarker; liquid biopsy; metabolome; ovarian cancer; proteome
    DOI:  https://doi.org/10.3390/cancers12113447
  14. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 17. pii: S1570-0232(20)31320-9. [Epub ahead of print]1161 122444
    Supercritical fluid chromatography - Mass spectrometry in metabolomics: Past, present, and future perspectives.
    Bas van de Velde, Davy Guillarme, Isabelle Kohler.
      Metabolomics, which consists of the comprehensive analysis of metabolites within a biological system, has been playing a growing role in the implementation of personalized medicine in modern healthcare. A wide range of analytical approaches are used in metabolomics, notably mass spectrometry (MS) combined to liquid chromatography (LC), gas chromatography (GC), or capillary electrophoresis (CE). However, none of these methods enable a comprehensive analysis of the metabolome, due to its extreme complexity and the large differences in physico-chemical properties between metabolite classes. In this context, supercritical fluid chromatography (SFC) represents a promising alternative approach to improve the metabolome coverage, while further increasing the analysis throughput. SFC, which uses supercritical CO2 as mobile phase, leads to numerous advantages such as improved kinetic performance and lower environmental impact. This chromatographic technique has gained a significant interest since the introduction of advanced instrumentation, together with the introduction of dedicated interfaces for hyphenating SFC to MS. Moreover, new developments in SFC column chemistry (including sub-2 µm particles), as well as the use of large amounts of organic modifiers and additives in the CO2-based mobile phase, significantly extended the application range of SFC, enabling the simultaneous analysis of a large diversity of metabolites. Over the last years, several applications have been reported in metabolomics using SFC-MS - from lipophilic compounds, such as steroids and other lipids, to highly polar compounds, such as carbohydrates, amino acids, or nucleosides. With all these advantages, SFC-MS is promised to a bright future in the field of metabolomics.
    Keywords:  Bioanalysis; Lipidomics; Mass spectrometry; Metabolomics; Supercritical fluid chromatography; Unified chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122444
  15. Mol Metab. 2020 Nov 18. pii: S2212-8778(20)30193-9. [Epub ahead of print] 101119
    Nuclear receptors and transcriptional regulation in non-alcoholic fatty liver disease.
    Yang Xiao, Mindy Kim, Mitchell A Lazar.
      BACKGROUND: As a result of a sedentary lifestyle and excess food consumption in modern society, non-alcoholic fatty liver disease (NAFLD) characterized by fat accumulation in the liver is becoming a major disease burden. Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD, characterized by inflammation and fibrosis which can lead to hepatocellular carcinoma and liver failure. Nuclear receptors (NRs) are a family of ligand-regulated transcription factors which closely control multiple aspects of metabolism, and their transcriptional activity is modulated by various ligands, including hormones and lipids. Thus, NRs serve as potential pharmacological targets for NAFLD/NASH and other metabolic diseases.SCOPE OF REVIEW: In this review, we provide a comprehensive overview of NRs that have been studied in the context of NAFLD/NASH with a focus on their transcriptional regulation, function in pre-clinical models, and studies of their clinical utility.
    MAJOR CONCLUSIONS: The transcriptional regulation of NRs is context-dependent. During the dynamic progression of NAFLD/NASH, NRs play diverse roles in multiple organs and different cell types in the liver, which highlights the necessity of targeting NRs in a stage-specific and cell-type specific manner to enhance the efficacy and safety of treatment methods.
    Keywords:  non-alcoholic fatty liver disease(NAFLD); nonalcoholic steatohepatitis(NASH); nuclear receptors(NRs); transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molmet.2020.101119
  16. Cell Metab. 2020 Nov 17. pii: S1550-4131(20)30595-7. [Epub ahead of print]
    The Power of Plasticity-Metabolic Regulation of Hepatic Stellate Cells.
    Parth Trivedi, Shuang Wang, Scott L Friedman.
      Hepatic stellate cells (HSCs) are resident non-parenchymal liver pericytes whose plasticity enables them to regulate a remarkable range of physiologic and pathologic responses. To support their functions in health and disease, HSCs engage pathways regulating carbohydrate, mitochondrial, lipid, and retinoid homeostasis. In chronic liver injury, HSCs drive hepatic fibrosis and are implicated in inflammation and cancer. To do so, the cells activate, or transdifferentiate, from a quiescent state into proliferative, motile myofibroblasts that secrete extracellular matrix, which demands rapid adaptation to meet a heightened energy need. Adaptations include reprogramming of central carbon metabolism, enhanced mitochondrial number and activity, endoplasmic reticulum stress, and liberation of free fatty acids through autophagy-dependent hydrolysis of retinyl esters that are stored in cytoplasmic droplets. As an archetype for pericytes in other tissues, recognition of the HSC's metabolic drivers and vulnerabilities offer the potential to target these pathways therapeutically to enhance parenchymal growth and modulate repair.
    DOI:  https://doi.org/10.1016/j.cmet.2020.10.026
  17. Nat Methods. 2020 Nov 23.
    Quantitative shotgun proteome analysis by direct infusion.
    Jesse G Meyer, Natalie M Niemi, David J Pagliarini, Joshua J Coon.
      Liquid chromatography-mass spectrometry (LC-MS) delivers sensitive peptide analysis for proteomics but requires extensive analysis time, reducing throughput. Here, we demonstrate that gas-phase peptide separation instead of LC enables fast proteome analysis. Using direct infusion-shotgun proteome analysis (DI-SPA) by data-independent acquisition mass spectrometry (DIA-MS), we demonstrate the targeted quantification of over 500 proteins within minutes of MS data collection (~3.5 proteins per second). We show the utility of this technology in performing a complex multifactorial proteomic study of interactions between nutrients, genotype and mitochondrial toxins in a collection of cultured human cells. More than 45,000 quantitative protein measurements from 132 samples were achieved in only ~4.4 h of MS data collection. Enabling fast, unbiased proteome quantification without LC, DI-SPA offers an approach to boost throughput, critical to drug and biomarker discovery studies that require analysis of thousands of proteomes.
    DOI:  https://doi.org/10.1038/s41592-020-00999-z
  18. EJC Suppl. 2020 Aug;15 38-48
    The influence of secreted factors and extracellular vesicles in ovarian cancer metastasis.
    Marta Hergueta-Redondo, Héctor Peinado.
      Ovarian cancer cells mainly metastasise within the peritoneal cavity, the lethal consequence of tumour progression in this cancer type. Classically, changes in tumour cells, such as epithelial to mesenchymal transition, involve the down-regulatinon of E-cadherin, activation of extracellular proteases and integrin-mediated adhesion. However, our current understanding of ovarian tumour progression suggests the implication of both intrinsic and extrinsic factors. It has been proposed that ovarian cancer metastases are a consequence of the crosstalk between cancer cells and the tumour microenvironment by soluble factors and extracellular vesicles. Characterisation of the alterations in both the tumour cells and the surrounding microenvironment has emerged as a new research field to understand ovarian cancer metastasis. In this mini review, we will summarise the most recent findings, focusing our attention on the role of secreted factors and extracellular vesicles in ovarian cancer metastasis.
    Keywords:  Cancer; Exosomes; Extracellular vesicles; Metastasis
    DOI:  https://doi.org/10.1016/j.ejcsup.2019.09.001
  19. Metabolites. 2020 Nov 19. pii: E472. [Epub ahead of print]10(11):
    Mouse Age Matters: How Age Affects the Murine Plasma Metabolome.
    Patrick Pann, Martin Hrabě de Angelis, Cornelia Prehn, Jerzy Adamski.
      A large part of metabolomics research relies on experiments involving mouse models, which are usually 6 to 20 weeks of age. However, in this age range mice undergo dramatic developmental changes. Even small age differences may lead to different metabolomes, which in turn could increase inter-sample variability and impair the reproducibility and comparability of metabolomics results. In order to learn more about the variability of the murine plasma metabolome, we analyzed male and female C57BL/6J, C57BL/6NTac, 129S1/SvImJ, and C3HeB/FeJ mice at 6, 10, 14, and 20 weeks of age, using targeted metabolomics (BIOCRATES AbsoluteIDQ™ p150 Kit). Our analysis revealed high variability of the murine plasma metabolome during adolescence and early adulthood. A general age range with minimal variability, and thus a stable metabolome, could not be identified. Age-related metabolomic changes as well as the metabolite profiles at specific ages differed markedly between mouse strains. This observation illustrates the fact that the developmental timing in mice is strain specific. We therefore stress the importance of deliberate strain choice, as well as consistency and precise documentation of animal age, in metabolomics studies.
    Keywords:  age; metabolism; metabolomics; mouse development; mouse strain
    DOI:  https://doi.org/10.3390/metabo10110472
  20. Front Oncol. 2020 ;10 554272
    Metabolic Compartmentalization at the Leading Edge of Metastatic Cancer Cells.
    Kara Wolfe, Ryo Kamata, Kester Coutinho, Takanari Inoue, Atsuo T Sasaki.
      Despite advances in targeted therapeutics and understanding in molecular mechanisms, metastasis remains a substantial obstacle for cancer treatment. Acquired genetic mutations and transcriptional changes can promote the spread of primary tumor cells to distant tissues. Additionally, recent studies have uncovered that metabolic reprogramming of cancer cells is tightly associated with cancer metastasis. However, whether intracellular metabolism is spatially and temporally regulated for cancer cell migration and invasion is understudied. In this review, we highlight the emergence of a concept, termed "membraneless metabolic compartmentalization," as one of the critical mechanisms that determines the metastatic capacity of cancer cells. In particular, we focus on the compartmentalization of purine nucleotide metabolism (e.g., ATP and GTP) at the leading edge of migrating cancer cells through the uniquely phase-separated microdomains where dynamic exchange of nucleotide metabolic enzymes takes place. We will discuss how future insights may usher in a novel class of therapeutics specifically targeting the metabolic compartmentalization that drives tumor metastasis.
    Keywords:  GTP-metabolism; cancer; leading edge; liquid-liquid phase separation; membraneless metabolic compartmentalization; metabolon; metastasis; purine biosynthesis
    DOI:  https://doi.org/10.3389/fonc.2020.554272
  21. Antioxidants (Basel). 2020 Nov 23. pii: E1169. [Epub ahead of print]9(11):
    Profiling of Low-Molecular-Weight Carbonyls and Protein Modifications in Flavored Milk.
    Michele Wölk, Theres Schröter, Ralf Hoffmann, Sanja Milkovska-Stamenova.
      Thermal treatments of dairy products favor oxidations, Maillard reactions, and the formation of sugar or lipid oxidation products. Additives including flavorings might enhance these reactions or even induce further reactions. Here we aimed to characterize protein modifications in four flavored milk drinks using samples along the production chain-raw milk, pasteurization, mixing with flavorings, heat treatment, and the commercial product. Therefore, milk samples were analyzed using a bottom up proteomics approach and a combination of data-independent (MSE) and data-dependent acquisition methods (DDA). Twenty-one small carbonylated lipids were identified by shotgun lipidomics triggering 13 protein modifications. Additionally, two Amadori products, 12 advanced glycation end products (AGEs), and 12 oxidation-related modifications were targeted at the protein level. The most common modifications were lactosylation, formylation, and carboxymethylation. The numbers and distribution of modification sites present in raw milk remained stable after pasteurization and mixing with flavorings, while the final heat treatment significantly increased lactosylation and hexosylation in qualitative and quantitative terms. The processing steps did not significantly affect the numbers of AGE-modified, oxidized/carbonylated, and lipid-carbonylated sites in proteins.
    Keywords:  Maillard reaction; flavored milk; low molecular weight carbonyls; oxidation/carbonylation; protein modifications
    DOI:  https://doi.org/10.3390/antiox9111169
  22. Cell Syst. 2020 Nov 18. pii: S2405-4712(20)30418-X. [Epub ahead of print]
    Multi-omics Analysis of CRISPRi-Knockdowns Identifies Mechanisms that Buffer Decreases of Enzymes in E. coli Metabolism.
    Stefano Donati, Michelle Kuntz, Vanessa Pahl, Niklas Farke, Dominik Beuter, Timo Glatter, José Vicente Gomes-Filho, Lennart Randau, Chun-Ying Wang, Hannes Link.
      Enzymes maintain metabolism, and their concentration affects cellular fitness: high enzyme levels are costly, and low enzyme levels can limit metabolic flux. Here, we used CRISPR interference (CRISPRi) to study the consequences of decreasing E. coli enzymes below wild-type levels. A pooled CRISPRi screen with 7,177 strains demonstrates that metabolism buffers fitness defects for hours after the induction of CRISPRi. We characterized the metabolome and proteome responses in 30 CRISPRi strains and elucidated three gene-specific buffering mechanisms: ornithine buffered the knockdown of carbamoyl phosphate synthetase (CarAB) by increasing CarAB activity, S-adenosylmethionine buffered the knockdown of homocysteine transmethylase (MetE) by de-repressing expression of the methionine pathway, and 6-phosphogluconate buffered the knockdown of 6-phosphogluconate dehydrogenase (Gnd) by activating a bypass. In total, this work demonstrates that CRISPRi screens can reveal global sources of metabolic robustness and identify local regulatory mechanisms that buffer decreases of specific enzymes. A record of this paper's transparent peer review process is included in the Supplemental Information.
    Keywords:  CRISPR interference; allosteric regulation; metabolic robustness; metabolomics; proteomics; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.cels.2020.10.011
  23. Bioessays. 2020 Dec;42(12): e2000052
    Metabolomics meets lipidomics: Assessing the small molecule component of metabolism.
    Hector Gallart-Ayala, Tony Teav, Julijana Ivanisevic.
      Metabolomics, including lipidomics, is emerging as a quantitative biology approach for the assessment of energy flow through metabolism and information flow through metabolic signaling; thus, providing novel insights into metabolism and its regulation, in health, healthy ageing and disease. In this forward-looking review we provide an overview on the origins of metabolomics, on its role in this postgenomic era of biochemistry and its application to investigate metabolite role and (bio)activity, from model systems to human population studies. We present the challenges inherent to this analytical science, and approaches and modes of analysis that are used to resolve, characterize and measure the infinite chemical diversity contained in the metabolome (including lipidome) of complex biological matrices. In the current outbreak of metabolic diseases such as cardiometabolic disorders, cancer and neurodegenerative diseases, metabolomics appears to be ideally situated for the investigation of disease pathophysiology from a metabolite perspective.
    Keywords:  energy metabolism; lipidomics; metabolic profiling; metabolic signaling; metabolite activity; metabolomics; quantitative biology approach
    DOI:  https://doi.org/10.1002/bies.202000052
  24. Front Cell Dev Biol. 2020 ;8 590192
    Dietary Energy Modulation and Autophagy: Exploiting Metabolic Vulnerabilities to Starve Cancer.
    Alyssa J Cozzo, Michael F Coleman, Jane B Pearce, Alexander J Pfeil, Suhas K Etigunta, Stephen D Hursting.
      Cancer cells experience unique and dynamic shifts in their metabolic function in order to survive, proliferate, and evade growth inhibition in the resource-scarce tumor microenvironment. Therefore, identification of pharmacological agents with potential to reprogram cancer cell metabolism may improve clinical outcomes in cancer therapy. Cancer cells also often exhibit an increased dependence on the process known as autophagy, both for baseline survival and as a response to stressors such as chemotherapy or a decline in nutrient availability. There is evidence to suggest that this increased dependence on autophagy in cancer cells may be exploitable clinically by combining autophagy modulators with existing chemotherapies. In light of the increased metabolic rate in cancer cells, interest is growing in approaches aimed at "starving" cancer through dietary and pharmacologic interventions that reduce availability of nutrients and pro-growth hormonal signals known to promote cancer progression. Several dietary approaches, including chronic calorie restriction and multiple forms of fasting, have been investigated for their potential anti-cancer benefits, yielding promising results in animal models. Induction of autophagy in response to dietary energy restriction may underlie some of the observed benefit. However, while interventions based on dietary energy restriction have demonstrated safety in clinical trials, uncertainty remains regarding translation to humans as well as feasibility of achieving compliance due to the potential discomfort and weight loss that accompanies dietary restriction. Further induction of autophagy through dietary or pharmacologic metabolic reprogramming interventions may enhance the efficacy of autophagy inhibition in the context of adjuvant or neo-adjuvant chemotherapy. Nonetheless, it remains unclear whether therapeutic agents aimed at autophagy induction, autophagy inhibition, or both are a viable therapeutic strategy for improving cancer outcomes. This review discusses the literature available for the therapeutic potential of these approaches.
    Keywords:  autophagy; caloric restriction; cancer; cancer therapy; fasting; metabolism
    DOI:  https://doi.org/10.3389/fcell.2020.590192
  25. Curr Protein Pept Sci. 2020 Nov 23.
    Characterization of Phosphorylated Proteins Using Mass Spectrometry.
    Li-Rong Yu, Timothy D Veenstra.
      Phosphorylation is arguably the most important post-translational modification that occurs within proteins. Phosphorylation is used as a signal to control numerous physiological activities ranging from gene expression to metabo-lism. Identifying phosphorylation sites within proteins was historically a challenge as it required either radioisotope label-ing or the use of phospho-specific antibodies. The advent of mass spectrometry (MS) has had a major impact on the abil-ity to qualitatively and quantitatively characterize phosphorylated proteins. In this article we describe MS methods for characterizing phosphorylation sites within individual proteins as well as entire proteome samples. The utility of these methods is illustrated in examples that show the information that can be gained using these MS techniques.
    Keywords:  Phosphorylation; mass spectrometry; peptide mapping; tandem mass spectrometry (MS2); phosphoproteomics; immobilized metal affinity chromatography (IMAC); metal oxide affinity chromatography (MOAC)
    DOI:  https://doi.org/10.2174/1389203721999201123200439
  26. Mol Cell Oncol. 2020 Sep 08. 7(6): 1805257
    DGAT1 protects tumor from lipotoxicity, emerging as a promising metabolic target for cancer therapy.
    Xiang Cheng, Feng Geng, Deliang Guo.
      We recently demonstrated that glioblastoma, the most lethal brain cancer, upregulates diacylglycerol O-acyltransferase 1 (DGAT1) to store excess fatty acids into triglycerides to prevent lipotoxicity and promote tumor growth. Targeting DGAT1 resulted in marked tumor cell death by triggering extensive oxidative stress, indicating that DGAT1 could be a promising target for cancer therapy.
    Keywords:  DGAT1; glioblastoma; lipotoxicity; oxidative stress; triglycerides
    DOI:  https://doi.org/10.1080/23723556.2020.1805257
  27. Food Res Int. 2020 Nov;pii: S0963-9969(20)30752-3. [Epub ahead of print]137 109727
    Comprehensive lipid analysis of green Arabica coffee beans by LC-HRMS/MS.
    Ana Carolina R Silva, Carol Cristine da Silva, Rafael Garrett, Claudia M Rezende.
      Lipids play an important role in coffee bean development, coffee brew and in the effects of coffee on human health. They account for around 17% of the dry bean weight and encompass different classes and subclasses, mostly triacylglycerols (TAG) and a minor quantity of phospholipids (PL) and βN-alkanoyl-5-hydroxytryptamides (C-5HT). To comprehensive profile these different lipids, it is important to evaluate extraction methods that provide high lipid coverage and to analyze the lipids in high-resolution techniques. In this work, liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was employed to comprehensive profile lipids from green Arabica coffee beans and to evaluate the extraction efficiency and lipid coverage of three methods: Bligh-Dyer (BD), Folch (FO), and Matyash (MA). The MA method yielded the greatest number of annotated compounds (131 lipids) compared to the other methods. In the positive electrospray ionization (ESI) mode, the main difference among extraction methods was observed for TAG and diacylglycerols, whereas for the negative ESI it was observed differences for phosphatidylinositol (PI), lysophosphatidylinositol and phosphatidic acid (p < 0.05). The analysis of coffees from different maturation stages and/or post-harvest processes were also performed using the MA method. Immature beans were discriminated from mature and overripe beans by its lower levels of C-5HT, PI, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylethanolamine. These results can help to better understand the coffee lipid composition and its association with coffee quality.
    Keywords:  Coffee; LC-MS; Lipidomics; Liquid-liquid extraction; MTBE; Orbitrap
    DOI:  https://doi.org/10.1016/j.foodres.2020.109727
  28. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 06. pii: S1570-0232(20)30059-3. [Epub ahead of print]1161 122371
    A simple LC-MS/MS method for the simultaneous quantification of endocannabinoids in biological samples.
    Maria Bobrich, Rico Schwarz, Robert Ramer, Philipp Borchert, Burkhard Hinz.
      The arachidonic acid derivatives N-arachidonoylethanolamine (anandamide; AEA), 2-arachidonoylglycerol (2-AG), N-arachidonoyldopamine (NADA), 2-arachidonoylglycerol ether (noladin ether; 2-AGE) and O-arachidonoylethanolamine (virodhamine; VA) were identified as physiological components of the endocannabinoid (EC) system. In order to gain further profound knowledge about the different EC-induced physiological and pathophysiological effects, appropriate analytical methods are required. The method described here uses liquid chromatography in combination with positive electrospray ionization mass spectrometry (LC-MS/MS) to quantify the concentrations of the above-mentioned EC compounds in cells. Sample preparation prior to LC-MS/MS analysis was performed by means of two liquid extractions with ethyl acetate. The method has been validated according to the bioanalytical guidelines of the Food and Drug Administration (FDA). The lower limits of quantification were 0.03 ng/mL for AEA, 2 ng/mL for 2-AG, 0.03 ng/mL for NADA, 0.3 ng/mL for 2-AGE and 0.15 ng/mL for VA. Linearity was demonstrated up to 10 ng/mL (AEA, NADA, 2-AGE and VA) and 50 ng/mL (2-AG). The values for intra- and inter-day precision and accuracy were within the guideline recommended acceptance criteria for assay validation. Low matrix effects and good recovery were found for AEA, 2-AG and 2-AGE, while a higher matrix effect was observed for NADA and VA. Extraction yields were lowest for VA. The method was used for EC measurement in different cell lines and in mouse brains.
    Keywords:  Endocannabinoids; Human cells; LC-MS/MS; Quantification; Validation
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122371
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