bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒09‒05
twenty-two papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh
  1. A Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer.
  2. A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system.
  3. c-MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency.
  4. Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for Putative Metabolite Annotation.
  5. CsoDIAq Software for Direct Infusion Shotgun Proteome Analysis.
  6. Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells.
  7. Metabolic perturbations in fibrosis disease.
  8. Extensive and Accurate Benchmarking of DIA Acquisition Methods and Software Tools Using a Complex Proteomic Standard.
  9. Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.
  10. Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology.
  11. Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry.
  12. Omega-3 Versus Omega-6 Fatty Acid Availability is Controlled by Hydrophobic Site Geometries of Phospholipase A2s.
  13. Development of a sensitive liquid chromatography-tandem mass spectrometry method for quantification of human plasma arginine vasopressin.
  14. Astrocyte metabolism of the medium-chain fatty acids octanoic acid and decanoic acid promotes GABA synthesis in neurons via elevated glutamine supply.
  15. CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors.
  16. Detecting lysosomal storage disorders by glycomic profiling using liquid chromatography mass spectrometry.
  17. From OCR and ECAR to energy: perspectives on the design and interpretation of bioenergetics studies.
  18. Lipid Metabolism.
  19. Parallel Derivatization Strategy for Comprehensive Profiling of Unconjugated and Glycine-conjugated Bile Acids Using: Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry.
  20. Patient-Derived Xenografts to Study Cancer Metabolism: When Does X Mark the Spot?
  21. Metabolomic Predictors of Non-alcoholic Steatohepatitis and Advanced Fibrosis in Children.
  22. SnapShot: Clinical proteomics.
  1. ACS Meas Sci Au. 2021 Aug 18. 1(1): 35-45
    A Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer.
    Michaela Schwaiger-Haber, Ethan Stancliffe, Valerie Arends, Bharat Thyagarajan, Miriam Sindelar, Gary J Patti.
      The thousands of features commonly observed when performing untargeted metabolomics with quadrupole time-of-flight (QTOF) and Orbitrap mass spectrometers often correspond to only a few hundred unique metabolites of biological origin, which is in the range of what can be assayed in a single targeted metabolomics experiment by using a triple quadrupole (QqQ) mass spectrometer. A major benefit of performing targeted metabolomics with QqQ mass spectrometry is the affordability of the instruments relative to high-resolution QTOF and Orbitrap platforms. Optimizing targeted methods to profile hundreds of metabolites on a QqQ mass spectrometer, however, has historically been limited by the availability of authentic standards, particularly for "unknowns" that have yet to be structurally identified. Here, we report a strategy to develop multiple reaction monitoring (MRM) methods for QqQ instruments on the basis of high-resolution spectra, thereby enabling us to use data from untargeted metabolomics to design targeted experiments without the need for authentic standards. We demonstrate that using high-resolution fragmentation data alone to design MRM methods results in the same quantitative performance as when methods are optimized by measuring authentic standards on QqQ instruments, as is conventionally done. The approach was validated by showing that Orbitrap ID-X data can be used to establish MRM methods on a Thermo TSQ Altis and two Agilent QqQs for hundreds of metabolites, including unknowns, without a dependence on standards. Finally, we highlight an application where metabolite profiling was performed on an ID-X and a QqQ by using the strategy introduced here, with both data sets yielding the same result. The described approach therefore allows us to use QqQ instruments, which are often associated with targeted metabolomics, to profile knowns and unknowns at a comprehensive scale that is typical of untargeted metabolomics.
    DOI:  https://doi.org/10.1021/acsmeasuresciau.1c00007
  2. Chem Phys Lipids. 2021 Aug 25. pii: S0009-3084(21)00078-5. [Epub ahead of print]240 105125
    A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system.
    Niels A W de Kok, Marten Exterkate, Ruben L H Andringa, Adriaan J Minnaard, Arnold J M Driessen.
      Simple, robust and versatile LC-MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC.
    Keywords:  Cardiolipin; Electrospray ionization mass spectrometry (ESI-MS); Lipidomics; Phospholipid; Reversed-phase liquid chromatography (RPLC); Tetraether lipid
    DOI:  https://doi.org/10.1016/j.chemphyslip.2021.105125
  3. Stem Cell Rev Rep. 2021 Sep 02.
    c-MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency.
    Javier Prieto, Juan Carlos García-Cañaveras, Marian León, Ramón Sendra, Xavier Ponsoda, Juan Carlos Izpisúa Belmonte, Agustín Lahoz, Josema Torres.
      Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency. c-MYC promotes anabolic metabolism, mitochondrial fitness and lipid remodelling early in cell reprogramming. A high rate of aerobic glycolysis is crucial to provide intermediaries for biosynthetic pathways. To ensure the availability of nucleotides, amino acids and lipids for cell proliferation, cells must provide with a constant flux of the elemental building blocks for macromolecule assembly and fulfil the anabolic demands to reach the critical cellular mass levels to satisfactorily undergo cell division. A high rate of aerobic glycolysis is induced by c-MYC, increasing the amounts of intracellular Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and glyceraldehyde-3-phosphate (GA3P), which can all enter pentose phosphate pathway (PPP) to produce Ribose-5-Phosphate (R5P) and NADPH, which are necessary for the biosynthesis of biomolecules such as proteins, nucleic acids, or lipids. C-MYC-dependent activation of glucose-6-phosphate dehydrogenase (G6PD) may play a critical role in the shunting of G6P to PPP and generation of NADPH. High glycolytic flux increases the amounts of dihydroxyacetone phosphate (DHAP), which is crucial for biosynthesis of phospholipids and triacylglycerols, and pyruvate (Pyr), which can be converted to citrate (Cit) in the mitochondria and enter the biosynthesis of fatty acids (FA). During cell reprogramming, c-MYC-dependent lipid remodelling leads to Polyunsaturated Fatty Acid (PUFA) downregulation and Monounsaturated Fatty Acid (MUFA) upregulation, which may play critical roles in cytoarchitectural remodelling of cell membrane or non-canonical autophagy, respectively. Cardiolipin (pink dots) rise early in cell reprogramming correlates with an increase in mitochondrial fitness, suggesting that c-MYC may restore proper levels of cardiolipins and antioxidant proteins, such as UCP2, to guarantee an optimal mitochondrial function while upholding ROS levels, reinforcing the idea of cell rejuvenation early in cell reprogramming.
    DOI:  https://doi.org/10.1007/s12015-021-10239-2
  4. Anal Chem. 2021 Aug 30.
    Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for Putative Metabolite Annotation.
    Danning Huang, Marcos Bouza, David A Gaul, Franklin E Leach, I Jonathan Amster, Frank C Schroeder, Arthur S Edison, Facundo M Fernández.
      Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass spectrometry (MS) are among the highest-performing analytical platforms used in metabolomics. Non-targeted metabolomics experiments, however, yield extremely complex datasets that make metabolite annotation very challenging and sometimes impossible. The high-resolution accurate mass measurements of the leading MS platforms greatly facilitate this process by reducing mass errors and spectral overlaps. When high resolution is combined with relative isotopic abundance (RIA) measurements, heuristic rules, and constraints during searches, the number of candidate elemental formula(s) can be significantly reduced. Here, we evaluate the performance of Orbitrap ID-X and 12T solariX FT-ICR mass spectrometers in terms of mass accuracy and RIA measurements and how these factors affect the assignment of the correct elemental formulas in the metabolite annotation pipeline. Quality of the mass measurements was evaluated under various experimental conditions (resolution: 120, 240, 500 K; automatic gain control: 5 × 104, 1 × 105, 5 × 105) for the Orbitrap MS platform. High average mass accuracy (<1 ppm for UPLC-Orbitrap MS and <0.2 ppm for direct infusion FT-ICR MS) was achieved and allowed the assignment of correct elemental formulas for over 90% (m/z 75-466) of the 104 investigated metabolites. 13C1 and 18O1 RIA measurements further improved annotation certainty by reducing the number of candidates. Overall, our study provides a systematic evaluation for two leading Fourier transform (FT)-based MS platforms utilized in metabolite annotation and provides the basis for applying these, individually or in combination, to metabolomics studies of biological systems.
    DOI:  https://doi.org/10.1021/acs.analchem.1c02224
  5. Anal Chem. 2021 Sep 01.
    CsoDIAq Software for Direct Infusion Shotgun Proteome Analysis.
    Caleb W Cranney, Jesse G Meyer.
      Direct infusion shotgun proteome analysis (DISPA) is a new paradigm for expedited mass spectrometry-based proteomics, but the original data analysis workflow was onerous. Here, we introduce CsoDIAq, a user-friendly software package for the identification and quantification of peptides and proteins from DISPA data. In addition to establishing a complete and automated analysis workflow with a graphical user interface, CsoDIAq introduces algorithmic concepts to spectrum-spectrum matching to improve peptide identification speed and sensitivity. These include spectra pooling to reduce search time complexity and a new spectrum-spectrum match score called match count and cosine, which improves target discrimination in a target-decoy analysis. Fragment mass tolerance correction also increased the number of peptide identifications. Finally, we adapt CsoDIAq to standard LC-MS DIA and show that it outperforms other spectrum-spectrum matching software.
    DOI:  https://doi.org/10.1021/acs.analchem.1c02021
  6. Expert Rev Proteomics. 2021 Sep 01.
    Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells.
    Jacqueline S Gerritsen, Forest M White.
      INTRODUCTION: Many pathologies, including cancer, have been associated with aberrant phosphorylation-mediated signaling networks that drive altered cell proliferation, migration, metabolic regulation, and can lead to systemic inflammation. Phosphoproteomics, the large-scale analysis of protein phosphorylation sites, has emerged as a powerful tool to define signaling network regulation and dysregulation in normal and pathological conditions.AREAS COVERED: We provide an overview of methodology for global phosphoproteomics as well as enrichment of specific subsets of the phosphoproteome, including phosphotyrosine and phospho-motif enrichment of kinase substrates. We review quantitative methods, advantages and limitations of different mass spectrometry acquisition formats, and computational approaches to extract biological insight from phosphoproteomics data. Throughout, we discuss various applications and their challenges in implementation.
    EXPERT OPINION: Over the past 20 years the field of phosphoproteomics has advanced to enable deep biological and clinical insight through the quantitative analysis of signaling networks. Future areas of development include Clinical Laboratory Improvement Amendments (CLIA)-approved methods for analysis of clinical samples, continued improvements in sensitivity to enable analysis of small numbers of rare cells and tissue microarrays, and computational methods to integrate data resulting from multiple systems-level quantitative analytical methods.
    Keywords:  cancer; cellular networks; kinase activity; mass spectrometry; molecular signaling; phosphoproteomics; phosphorylation
    DOI:  https://doi.org/10.1080/14789450.2021.1976152
  7. Int J Biochem Cell Biol. 2021 Aug 27. pii: S1357-2725(21)00153-9. [Epub ahead of print] 106073
    Metabolic perturbations in fibrosis disease.
    Chuin Ying Ung, Alexandros Onoufriadis, Maddy Parsons, John A McGrath, Tanya J Shaw.
      Metabolic changes occur in all forms of disease but their impact on fibrosis is a relatively recent area of interest. This review provides an overview of the major metabolic pathways, glycolysis, amino acid metabolism and lipid metabolism, and highlights how they influence fibrosis at a cellular and tissue level, drawing on key discoveries in dermal, renal, pulmonary and hepatic fibrosis. The emerging influence of adipose tissue-derived cytokines is discussed and brings a link between fibrosis and systemic metabolism. To close, the concept of targeting metabolism for fibrotic therapy is reviewed, drawing on lessons from the more established field of cancer metabolism, with an emphasis on important considerations for clinical translation.
    Keywords:  Fibrosis; adipokines; fibroblasts; metabolism; therapy
    DOI:  https://doi.org/10.1016/j.biocel.2021.106073
  8. J Proteome Res. 2021 Sep 02.
    Extensive and Accurate Benchmarking of DIA Acquisition Methods and Software Tools Using a Complex Proteomic Standard.
    Clarisse Gotti, Florence Roux-Dalvai, Charles Joly-Beauparlant, Loïc Mangnier, Mickaël Leclercq, Arnaud Droit.
      Over the past decade, the data-independent acquisition mode has gained popularity for broad coverage of complex proteomes by LC-MS/MS and quantification of low-abundance proteins. However, there is no consensus in the literature on the best data acquisition parameters and processing tools to use for this specific application. Here, we present the most comprehensive comparison of DIA workflows on Orbitrap instruments published so far in the field of proteomics. Using a standard human 48 proteins mixture (UPS1-Sigma) at 8 different concentrations in an E. coli proteome background, we tested 36 workflows including 4 different DIA window acquisition schemes and 6 different software tools (DIA-NN, DIA-Umpire, OpenSWATH, ScaffoldDIA, Skyline, and Spectronaut) with or without the use of a DDA spectral library. On the basis of the number of proteins identified, quantification linearity and reproducibility, as well as sensitivity and specificity in 28 pairwise comparisons of different UPS1 concentrations, we summarize the major considerations and propose guidelines for choosing the DIA workflow best suited for LC-MS/MS proteomic analyses. Our 96 DIA raw files and software outputs have been deposited on ProteomeXchange for testing or developing new DIA processing tools.
    Keywords:  data-independent acquisition; label-free quantification; proteomic standard; software benchmarking
    DOI:  https://doi.org/10.1021/acs.jproteome.1c00490
  9. Nat Commun. 2021 Sep 01. 12(1): 5214
    Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.
    Arthur C Sletten, Jessica W Davidson, Busra Yagabasan, Samantha Moores, Michaela Schwaiger-Haber, Hideji Fujiwara, Sarah Gale, Xuntian Jiang, Rohini Sidhu, Susan Gelman, Shuang Zhao, Gary Patti, Daniel S Ory, Jean E Schaffer.
      Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.
    DOI:  https://doi.org/10.1038/s41467-021-25457-y
  10. Crit Rev Clin Lab Sci. 2021 Sep 02. 1-18
    Quantifying RNA modifications by mass spectrometry: a novel source of biomarkers in oncology.
    Amandine Amalric, Amandine Bastide, Aurore Attina, Armelle Choquet, Jerome Vialaret, Sylvain Lehmann, Alexandre David, Christophe Hirtz.
      Despite significant progress in targeted therapies, cancer recurrence remains a major cause of mortality worldwide. Identification of accurate biomarkers, through molecular profiling in healthy and cancer patient samples, will improve diagnosis and promote personalized medicine. While genetic and epigenetic alterations of DNA are currently exploited as cancer biomarkers, their robustness is limited by tumor heterogeneity. Recently, cancer-associated changes in RNA marks have emerged as a promising source of diagnostic and prognostic biomarkers. RNA epigenetics (also known as epitranscriptomics) is an emerging field in which at least 150 chemical modifications in all types of RNA (mRNA, tRNA, lncRNA, rRNA, and microRNA) have been detected. These modifications fine-tune gene expression in both physiological and pathological processes. A growing number of studies have established links between specific modified nucleoside levels in solid/liquid biopsies, and cancer onset and progression. In this review, we highlight the potential role of epitranscriptomic markers in refining cancer diagnosis and/or prognosis. RNA modification patterns may contain important information for establishing an initial diagnosis, monitoring disease evolution, and predicting response to treatment. Furthermore, recent developments in mass spectrometry allow reliable quantification of RNA marks in solid biopsies and biological fluids. We discuss the great potential of mass spectrometry for identifying epitranscriptomic biomarker signatures in cancer diagnosis. While there are various methods to quantify modified nucleosides, most are unable to detect and quantify more than one type of RNA modification at a time. Mass spectrometry analyses, especially GC-MS/MS and LC-MS/MS, overcome this limitation and simultaneously detect modified nucleosides by multiple reaction monitoring. Indeed, several groups are currently validating mass spectrometry methods that quantify several nucleosides at one time in liquid biopsies. The challenge now is to exploit these powerful analytical tools to establish epitranscriptomic signatures that should open new perspectives in personalized medicine. This review summarizes the growing clinical field of analysis of RNA modifications and discusses pre-analytical and analytical approaches, focusing in particular on the development of new mass spectrometry tools and their clinical applications.
    Keywords:  Epitranscriptomics; biomarker; cancer; mass spectrometry
    DOI:  https://doi.org/10.1080/10408363.2021.1958743
  11. J Vis Exp. 2021 Aug 13.
    Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry.
    Danye Qiu, Verena B Eisenbeis, Adolfo Saiardi, Henning J Jessen.
      Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.
    DOI:  https://doi.org/10.3791/62847
  12. J Lipid Res. 2021 Aug 30. pii: S0022-2275(21)00095-X. [Epub ahead of print] 100113
    Omega-3 Versus Omega-6 Fatty Acid Availability is Controlled by Hydrophobic Site Geometries of Phospholipase A2s.
    Daiki Hayashi, Varnavas D Mouchlis, Edward A Dennis.
      Human phospholipase A2's (PLA2) constitute a superfamily of enzymes that hydrolyze the sn-2 acyl-chain of glycerophospholipids, producing lysophospholipids and free fatty acids. Each PLA2 enzyme type contributes to specific biological functions based on its expression, subcellular localization, and substrate specificity. Among the PLA2 superfamily, the cytosolic cPLA2 enzymes, calcium-independent iPLA2 enzymes, and secreted sPLA2 enzymes are implicated in many diseases, but a central issue is the preference for double-bond positions in polyunsaturated fatty acids (PUFAs) occupying the sn-2 position of membrane phospholipids. We demonstrate that each PLA2 has a unique preference between the specific omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 arachidonic acid (AA), which are the precursors of most pro-inflammatory and anti-inflammatory or resolving eicosanoids and related oxylipins. Surprisingly, we discovered that human cPLA2 selectively prefers AA, whereas iPLA2 prefers EPA, and sPLA2 prefers DHA as substrate. We determined the optimal binding of each phospholipid substrate in the active site of each PLA2 to explain these specificities. To investigate this, we utilized recently-developed lipidomics-based LC-MS/MS and GC/MS assays to determine the sn-2 acyl chain specificity in mixtures of phospholipids. We performed μs timescale molecular dynamics (MD) simulations to reveal unique active site properties, especially how the precise hydrophobic cavity accommodation of the sn-2 acyl chain contributes to the stability of substrate binding and the specificity of each PLA2 for AA, EPA, or DHA. This study provides the first comprehensive picture of the unique substrate selectivity of each PLA2 for omega-3 and omega-6 fatty acids.
    Keywords:  AA; DHA; EPA; arachidonic acid; fatty acid; glycerophospholipid; omega-3; omega-6; phospholipase A2; substrate specificity
    DOI:  https://doi.org/10.1016/j.jlr.2021.100113
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Aug 23. pii: S1570-0232(21)00384-6. [Epub ahead of print]1181 122903
    Development of a sensitive liquid chromatography-tandem mass spectrometry method for quantification of human plasma arginine vasopressin.
    Masaki Takiwaki, Fumio Nomura, Mamoru Satoh, Sachio Tsuchida, Kazuo Otake, Junko Takagi.
      BACKGROUND AND AIMS: Direct measurement of arginine vasopressin (AVP) via immunoassays is not widely conducted, mainly because of technical constraints. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been widely used as the gold standard in clinical chemistry. Here, we aimed to develop an MS-based assay to determine human plasma AVP and compare the results with those obtained using a conventional immunoassay.MATERIALS AND METHODS: We developed a protocol using triple quadrupole MS coupled with LC for the measurement of human plasma AVP. Analytical evaluations of the method were performed, and the results obtained using LC/MS/MS and radioimmunoassay (RIA) were compared.
    RESULTS: The lower limit of quantification (LLOQ) for plasma AVP obtained using LC/MS/MS and RIA were 0.2 and 0.4 pg/mL, respectively. Although there was a weak overall correlation between the results obtained using the two different methods, the RIA results did not agree with the LC/MS/MS results, particularly at low concentrations.
    CONCLUSIONS: AVP detection through RIA is not satisfactory compared with that using LC/MS/MS. Diagnostic values of direct AVP measurements must be evaluated based on the results obtained via sensitive and accurate MS-based methods rather than those obtained through RIA.
    Keywords:  Arginine vasopressin; Liquid chromatography-tandem mass spectrometry; Radioimmunoassay
    DOI:  https://doi.org/10.1016/j.jchromb.2021.122903
  14. Mol Brain. 2021 Sep 03. 14(1): 132
    Astrocyte metabolism of the medium-chain fatty acids octanoic acid and decanoic acid promotes GABA synthesis in neurons via elevated glutamine supply.
    Jens V Andersen, Emil W Westi, Emil Jakobsen, Nerea Urruticoechea, Karin Borges, Blanca I Aldana.
      The medium-chain fatty acids octanoic acid (C8) and decanoic acid (C10) are gaining attention as beneficial brain fuels in several neurological disorders. The protective effects of C8 and C10 have been proposed to be driven by hepatic production of ketone bodies. However, plasma ketone levels correlates poorly with the cerebral effects of C8 and C10, suggesting that additional mechanism are in place. Here we investigated cellular C8 and C10 metabolism in the brain and explored how the protective effects of C8 and C10 may be linked to cellular metabolism. Using dynamic isotope labeling, with [U-13C]C8 and [U-13C]C10 as metabolic substrates, we show that both C8 and C10 are oxidatively metabolized in mouse brain slices. The 13C enrichment from metabolism of [U-13C]C8 and [U-13C]C10 was particularly prominent in glutamine, suggesting that C8 and C10 metabolism primarily occurs in astrocytes. This finding was corroborated in cultured astrocytes in which C8 increased the respiration linked to ATP production, whereas C10 elevated the mitochondrial proton leak. When C8 and C10 were provided together as metabolic substrates in brain slices, metabolism of C10 was predominant over that of C8. Furthermore, metabolism of both [U-13C]C8 and [U-13C]C10 was unaffected by etomoxir indicating that it is independent of carnitine palmitoyltransferase I (CPT-1). Finally, we show that inhibition of glutamine synthesis selectively reduced 13C accumulation in GABA from [U-13C]C8 and [U-13C]C10 metabolism in brain slices, demonstrating that the glutamine generated from astrocyte C8 and C10 metabolism is utilized for neuronal GABA synthesis. Collectively, the results show that cerebral C8 and C10 metabolism is linked to the metabolic coupling of neurons and astrocytes, which may serve as a protective metabolic mechanism of C8 and C10 supplementation in neurological disorders.
    Keywords:  Capric acid (C10); Caprylic acid (C8); MCFA; MCT; Mitochondria; Neurotransmitter recycling; β-hydroxybutyrate
    DOI:  https://doi.org/10.1186/s13041-021-00842-2
  15. Anal Chem. 2021 Aug 30.
    CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors.
    Ren-Qi Wang, Jun Ding, Ye Geng, Yuan-Zheng Li, Ying-Wu Mei, Kai Bao, Huai-Dong Yu, Yu-Qi Feng.
      Sequential window acquisition of all theoretical spectra (SWATH) as a typical data-independent acquisition (DIA) strategy is favorable for untargeted metabolomics. It could theoretically acquire product ions of all precursor ions, including precursor ions showing chromatographic peaks of rather poor qualities. However, existing data processing methods present limited capabilities in capturing poor-quality peaks of precursor ions. Thus, although their product ions could be acquired, their precursor ions are absent. Here, we present a new strategy, chromatographic retention behavior-SWATH (CRB-SWATH), that could unbiasedly capture poor-quality peaks and provide high resolutions of multiplexed mass spectroscopy (MS/MS) spectra in SWATH datasets. CRB-SWATH monitors CRBs of SWATH-MS signals under a series of altered elution gradients. As signals of compounds differ from noise by showing CRBs, both the precursor and fragment ions are captured, while ignoring their peak qualities. Moreover, CRB-SWATH offers good chances to resolve highly multiplexed MS/MS spectra in SWATH datasets because precursor ions coeluted in a single elution gradient often present different CRBs. In the untargeted metabolic analysis of Hela cell extracts, CRB-SWATH showed the advantage in exclusively capturing 2645 ions of poor-quality peaks (i.e., tiny peaks, discontinuous ion traces, tailing peaks, zigzag peaks, etc.), accounting for 34.4% of all the untargeted precursor ions extracted. Therein, it is noteworthy that among 2116 negative ions detected in hydrophilic interaction liquid chromatography (HILIC) mode, 1284 poor-quality ion peaks (>60%) were exclusively captured by CRB-SWATH. As CRB-SWATH automatically captures a large sum of true ion peaks of poor qualities, extracts MS/MS spectra of high purities, and provides chromatographic retention behaviors of untargeted metabolites for identification and classification, it could be a useful metabolomics tool for understanding biological phenomena better.
    DOI:  https://doi.org/10.1021/acs.analchem.1c01841
  16. Mol Genet Metab. 2021 Aug 21. pii: S1096-7192(21)00770-8. [Epub ahead of print]
    Detecting lysosomal storage disorders by glycomic profiling using liquid chromatography mass spectrometry.
    Justin Mak, Tina M Cowan.
      BACKGROUND: Urine and plasma biomarker testing for lysosomal storage disorders by liquid chromatography mass spectrometry (LC-MS) currently requires multiple analytical methods to detect the abnormal accumulation of oligosaccharides, mucopolysaccharides, and glycolipids. To improve clinical testing efficiency, we developed a single LC-MS method to simultaneously identify disorders of oligosaccharide, mucopolysaccharide, and glycolipid metabolism with minimal sample preparation.METHODS: We created a single chromatographic method for separating free glycans and glycolipids in their native form, using an amide column and high pH conditions. We used this glycomic profiling method both in untargeted analyses of patient and control urines using LC ion-mobility high-resolution MS (biomarker discovery), and targeted analyses of urine, serum, and dried blood spot samples by LC-MS/MS (clinical validation).
    RESULTS: Untargeted glycomic profiling revealed twenty biomarkers that could identify and subtype mucopolysaccharidoses. We incorporated these with known oligosaccharide and glycolipid biomarkers into a rapid test that identifies at least 27 lysosomal storage disorders, including oligosaccharidoses, mucopolysaccharidoses, sphingolipidoses, glycogen storage disorders, and congenital disorders of glycosylation and de-glycosylation. In a validation set containing 115 urine samples from patients with lysosomal storage disorders, all were unambiguously distinguished from normal controls, with correct disease subtyping for 88% (101/115) of cases. Glucosylsphingosine was reliably elevated in dried blood spots from Gaucher disease patients with baseline resolution from galactosylsphingosine.
    CONCLUSION: Glycomic profiling by liquid chromatography mass spectrometry identifies a range of lysosomal storage disorders. This test can be used in clinical evaluations to rapidly focus a diagnosis, as well as to clarify or support additional gene sequencing and enzyme studies.
    Keywords:  Biomarker discovery; Glycomics; Inborn errors of metabolism; Liquid chromatography mass spectrometry; Lysosomal storage disorders; Metabolomics
    DOI:  https://doi.org/10.1016/j.ymgme.2021.08.006
  17. J Biol Chem. 2021 Aug 27. pii: S0021-9258(21)00941-8. [Epub ahead of print] 101140
    From OCR and ECAR to energy: perspectives on the design and interpretation of bioenergetics studies.
    Cameron A Schmidt, Kelsey H Fisher-Wellman, P Darrell Neufer.
      Biological energy transduction underlies all physiological phenomena in cells. The metabolic systems that support energy transduction have been of great interest due to their association with numerous pathologies including diabetes, cancer, rare genetic diseases, and aberrant cell death. Commercially available bioenergetics technologies (e.g. extracellular flux analysis, high resolution respirometry, fluorescent dye kits, etc.) have made practical assessment of metabolic parameters widely accessible. This has facilitated an explosion in the number of studies exploring, in particular, the biological implications of oxygen consumption rate (OCR) and substrate level phosphorylation via glycolysis (i.e. via extracellular acidification rate (ECAR)). Though these technologies have demonstrated substantial utility and broad applicability to cell biology research, they are also susceptible to historical assumptions, experimental limitations, and other caveats that have led to premature and/or erroneous interpretations. This review enumerates various important considerations for designing and interpreting cellular and mitochondrial bioenergetics experiments, some common challenges and pitfalls in data interpretation, and some potential 'next steps' to be taken that can address these highlighted challenges.
    Keywords:  ATP; Anaerobic glycolysis; Bioenergetics; Cell metabolism; Mitochondria
    DOI:  https://doi.org/10.1016/j.jbc.2021.101140
  18. Cold Spring Harb Perspect Biol. 2021 Sep 01. pii: a040576. [Epub ahead of print]13(9):
    Lipid Metabolism.
    Navdeep S Chandel.
      
    DOI:  https://doi.org/10.1101/cshperspect.a040576
  19. J Steroid Biochem Mol Biol. 2021 Aug 25. pii: S0960-0760(21)00179-5. [Epub ahead of print] 105986
    Parallel Derivatization Strategy for Comprehensive Profiling of Unconjugated and Glycine-conjugated Bile Acids Using: Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry.
    Ting Hu, Han Li, Benshan Xu, Ping Du, Lihong Liu, Zhuoling An.
      Bile acids (BAs) are steroidal compounds that play important roles in the occurrence and development of liver injury. However, comprehensive characterization of BAs was rarely reported due to the limitations of both standards access and detection sensitivity. In this study, a parallel derivatization strategy was established for the sensitive and comprehensive profiling of unconjugated and glycine-conjugated BAs by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Two structural analogues 2-hydrazinyl-4,6-dimethylpyrimidine (DMP) and 2-hydrazinylpyrimidine (DP) were used as the parallel derivatization reagents for BAs labeling, facilitating the improvements of both detection sensitivities and chromatographic performances. The derivatization reactions can be completed in 20 min at room temperature, with derivatization efficacy higher than 99%. Through derivatization, the sensitivity of BAs increased dozens or hundreds of times compared to their non-derivatized forms. Due to the structural similarities of derivatized BAs, general MS parameters can be forged for the analysis of DMP and DP labeled BAs. In addition, the DP labeled BAs were incorporated into the DMP derivatized biological samples for both the discovery and comprehensive characterization of BAs. Retention time shift (RTS) and peak area ratio (PAR) induced by the parallel DMP and DP labeled BAs were used for the rapid identification of BAs from complex biological samples. More than 200 BAs were profiled in rat serum using this parallel derivatization strategy. Further, the new strategy was successfully implemented in BAs profiling of serum samples from tripterysium glycosides-induced liver injury rat model. The disturbance of the BA metabolism network was further interpreted.
    Keywords:  UHPLC-MS/MS; bile acid; derivatization; drug-induced liver injury; tripterysium glycosides
    DOI:  https://doi.org/10.1016/j.jsbmb.2021.105986
  20. Cancer Res. 2021 Sep 01. 81(17): 4399-4401
    Patient-Derived Xenografts to Study Cancer Metabolism: When Does X Mark the Spot?
    Christopher S Nabel, Matthew G Vander Heiden.
      A major goal of cancer research is to understand the requirements for cancer growth and progression that can be exploited to treat patients. Model systems reduce the complexity and heterogeneity of human cancers to explore therapeutic hypotheses, however, some relevant aspects of human biology are not well represented by certain models, complicating the translation of preclinical findings to help patients. Here we discuss the advantages and limitations of patient-derived xenografts as a model system to study cancer metabolism, offering a framework to best use these models to address different types of metabolism-specific research questions.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0770
  21. Front Microbiol. 2021 ;12 713234
    Metabolomic Predictors of Non-alcoholic Steatohepatitis and Advanced Fibrosis in Children.
    Kattayoun Kordy, Fan Li, David J Lee, Jason M Kinchen, Michael H Jew, Maria Eduarda La Rocque, Sara Zabih, Monica Saavedra, Cora Woodward, Nicole J Cunningham, Nicole H Tobin, Grace M Aldrovandi.
      Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in western countries both in children and adults. Metabolic dysregulation associated with gut microbial dysbiosis may influence disease progression from hepatic steatosis to inflammation and subsequent fibrosis. Using a multi-omics approach, we profiled the oral and fecal microbiome and plasma metabolites from 241 predominantly Latino children with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), and controls. Children with more severe liver pathology were dysbiotic and had increased gene content associated with lipopolysaccharide biosynthesis and lipid, amino acid and carbohydrate metabolism. These changes were driven by increases in Bacteroides and concomitant decreases of Akkermansia, Anaerococcus, Corynebacterium, and Finegoldia. Non-targeted mass spectrometry revealed perturbations in one-carbon metabolism, mitochondrial dysfunction, and increased oxidative stress in children with steatohepatitis and fibrosis. Random forests modeling of plasma metabolites was highly predictive of non-alcoholic steatohepatitis (NASH) (97% accuracy) and hepatic fibrosis, steatosis and lobular inflammation (93.8% accuracy), and can differentiate steatohepatitis from simple steatosis (90.0% accuracy). Multi-omics predictive models for disease and histology findings revealed perturbations in one-carbon metabolism, mitochondrial dysfunction, and increased oxidative stress in children with steatohepatitis and fibrosis. These results highlight the promise of non-invasive biomarkers for the growing epidemic of fatty liver disease.
    Keywords:  NAFLD; NASH; microbiome; multiomics; pediatrics
    DOI:  https://doi.org/10.3389/fmicb.2021.713234
  22. Cell. 2021 Sep 02. pii: S0092-8674(21)00985-5. [Epub ahead of print]184(18): 4840-4840.e1
    SnapShot: Clinical proteomics.
    Yi Zhu, Ruedi Aebersold, Matthias Mann, Tiannan Guo.
      In-depth quantitative proteomic analysis of clinical specimens offers unique and invaluable insights into their protein composition and biochemical state in physiological and pathological conditions. A plethora of methodologies have been developed and customized. Here, we summarize the most common and emerging mass spectrometry (MS)-based workflows. Due to space limitations the overview cannot be complete. To view this SnapShot, open or download the PDF.
    DOI:  https://doi.org/10.1016/j.cell.2021.08.015
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