bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2023‒03‒12
24 papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh
  1. MAW: the reproducible Metabolome Annotation Workflow for untargeted tandem mass spectrometry.
  2. What can we learn from the platelet lipidome?
  3. Making single-cell proteomics biologically relevant.
  4. Visible-Light Paternò-Büchi Reaction for Lipidomic Profiling at Detailed Structure Levels.
  5. ASS1-mediated reductive carboxylation of cytosolic glutamine confers ferroptosis resistance in cancer cells.
  6. Optimized plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial.
  7. Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects.
  8. Targeting GPX4 in human cancer: Implications of ferroptosis induction for tackling cancer resilience.
  9. Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.
  10. Polyunsaturated Fatty Acids Drive Lipid Peroxidation during Ferroptosis.
  11. Simple In Vitro 18O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.
  12. A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation.
  13. Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells.
  14. Editorial: Cancer cell reprogramming: Impact on carcinogenesis and cancer progression.
  15. High-throughput site-specific N-glycoproteomics reveals glyco-signatures for liver disease diagnosis.
  16. Sequential isolation of metabolites and lipids from a single sample to achieve multiomics by using TRIzol reagent.
  17. Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension.
  18. Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.
  19. Bioorthogonal Peptide Enrichment From Complex Samples Using a Rink-Amide-Based Catch-and-Release Strategy.
  20. Lipidome of the Bacteroides Genus Containing New Peptidolipid and Sphingolipid Families Revealed by Multiple-Stage Mass Spectrometry.
  21. Editorial: Targeting cancer cell intracellular metabolism as a strategy against therapy resistance.
  22. Characterization of Dendritic Cell Metabolism by Flow Cytometry.
  23. Lactate in the tumor microenvironment: A rising star for targeted tumor therapy.
  24. Mass spectrometry imaging: the rise of spatially resolved single-cell omics.
  1. J Cheminform. 2023 Mar 04. 15(1): 32
    MAW: the reproducible Metabolome Annotation Workflow for untargeted tandem mass spectrometry.
    Mahnoor Zulfiqar, Luiz Gadelha, Christoph Steinbeck, Maria Sorokina, Kristian Peters.
      Mapping the chemical space of compounds to chemical structures remains a challenge in metabolomics. Despite the advancements in untargeted liquid chromatography-mass spectrometry (LC-MS) to achieve a high-throughput profile of metabolites from complex biological resources, only a small fraction of these metabolites can be annotated with confidence. Many novel computational methods and tools have been developed to enable chemical structure annotation to known and unknown compounds such as in silico generated spectra and molecular networking. Here, we present an automated and reproducible Metabolome Annotation Workflow (MAW) for untargeted metabolomics data to further facilitate and automate the complex annotation by combining tandem mass spectrometry (MS2) input data pre-processing, spectral and compound database matching with computational classification, and in silico annotation. MAW takes the LC-MS2 spectra as input and generates a list of putative candidates from spectral and compound databases. The databases are integrated via the R package Spectra and the metabolite annotation tool SIRIUS as part of the R segment of the workflow (MAW-R). The final candidate selection is performed using the cheminformatics tool RDKit in the Python segment (MAW-Py). Furthermore, each feature is assigned a chemical structure and can be imported to a chemical structure similarity network. MAW is following the FAIR (Findable, Accessible, Interoperable, Reusable) principles and has been made available as the docker images, maw-r and maw-py. The source code and documentation are available on GitHub ( https://github.com/zmahnoor14/MAW ). The performance of MAW is evaluated on two case studies. MAW can improve candidate ranking by integrating spectral databases with annotation tools like SIRIUS which contributes to an efficient candidate selection procedure. The results from MAW are also reproducible and traceable, compliant with the FAIR guidelines. Taken together, MAW could greatly facilitate automated metabolite characterization in diverse fields such as clinical metabolomics and natural product discovery.
    Keywords:  FAIR; Metabolite annotation; Tandem mass spectrometry; Untargeted metabolomics; Workflow
    DOI:  https://doi.org/10.1186/s13321-023-00695-y
  2. Platelets. 2023 Dec;34(1): 2182180
    What can we learn from the platelet lipidome?
    Gaëtan Chicanne, Jean Darcourt, Justine Bertrand-Michel, Cédric Garcia, Agnès Ribes, Bernard Payrastre.
      Besides their proteome, platelets use, in all responses to the environmental cues, a huge and diverse family of hydrophobic and amphipathic small molecules involved in structural, metabolic and signaling functions; the lipids. Studying how platelet lipidome changes modulate platelet function is an old story constantly renewed through the impressive technical advances allowing the discovery of new lipids, functions and metabolic pathways. Technical progress in analytical lipidomic profiling by top-of-the-line approaches such as nuclear magnetic resonance and gas chromatography or liquid chromatography coupled to mass spectrometry enables either large-scale analysis of lipids or targeted lipidomics. With the support of bioinformatics tools and databases, it is now possible to investigate thousands of lipids over a concentration range of several orders of magnitude. The lipidomic landscape of platelets is considered a treasure trove, not only able to expand our knowledge of platelet biology and pathologies but also to bring diagnostic and therapeutic opportunities. The aim of this commentary article is to summarize the advances in the field and to highlight what lipidomics can tell us about platelet biology and pathophysiology.
    Keywords:  Lipidomics; platelet
    DOI:  https://doi.org/10.1080/09537104.2023.2182180
  3. Nat Methods. 2023 Mar;20(3): 320-323
    Making single-cell proteomics biologically relevant.
    Florian A Rosenberger, Marvin Thielert, Matthias Mann.
      
    DOI:  https://doi.org/10.1038/s41592-023-01771-9
  4. Anal Chem. 2023 Mar 10.
    Visible-Light Paternò-Büchi Reaction for Lipidomic Profiling at Detailed Structure Levels.
    Hengxue Shi, Zhenshu Tan, Xiangyu Guo, Hanlin Ren, Shengzhuo Wang, Yu Xia.
      The Paternò-Büchi (PB) derivatization of carbon-carbon double bond (C═C) has been increasingly employed with tandem mass spectrometry to analyze unsaturated lipids. It enables the discovery of altered or uncanonical lipid desaturation metabolism, which would be otherwise undetected by conventional methods. Although highly useful, the reported PB reactions only provide moderate yield (∼30%). Herein, we aim to determine the key factors that affect the PB reactions and develop a system with improved capabilities for lipidomic analysis. An Ir(III) photocatalyst is chosen as the triplet energy donor for the PB reagent under 405 nm light irradiation, while phenylglyoxalate and its charge-tagging version, pyridylglyoxalate, are developed as the most efficient PB reagents. The above visible-light PB reaction system provides higher PB conversions than all previously reported PB reactions. Around 90% conversion can be achieved at high concentrations (>0.5 mM) for different classes of lipids but drops as the lipid concentration decreases. The visible-light PB reaction has then been integrated with shotgun and liquid chromatography-based workflows. The limits of detection for locating C═C in standard lipids of glycerophospholipids (GPLs) and triacylglycerides (TGs) are in the sub-nM to nM range. More than 600 distinct GPLs and TGs have been profiled at the C═C location level or the sn-position level from the total lipid extract of bovine liver, demonstrating that the developed method is capable of large-scale lipidomic analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.3c00085
  5. Cancer Res. 2023 Mar 09. pii: CAN-22-1999. [Epub ahead of print]
    ASS1-mediated reductive carboxylation of cytosolic glutamine confers ferroptosis resistance in cancer cells.
    Qiangsheng Hu, Jie Dai, Zheng Zhang, Huansha Yu, Jing Zhang, Xinsheng Zhu, Yi Qin, Lele Zhang, Peng Zhang.
      Induction of ferroptosis, a recently defined form of nonapoptotic cell death caused by iron-dependent lipid peroxidation, has emerged as an anti-cancer strategy. Erastin is a ferroptosis activator that promotes cell death that not only depends on the depletion of cellular cysteine but also relies on mitochondrial oxidative metabolism of glutamine. Here, we demonstrate that ASS1, a key enzyme involved in the urea cycle, plays a crucial role in ferroptosis resistance. Loss of ASS1 increased the sensitivity of non-small cell lung cancer (NSCLC) cells to erastin in vitro and decreased tumor growth in vivo. Metabolomics analysis with stable isotope-labeled glutamine showed that ASS1 promotes reductive carboxylation of cytosolic glutamine and compromises the oxidative TCA cycle from glutamine anaplerosis, reducing mitochondrial-derived lipid reactive oxygen species. Moreover, transcriptome sequencing showed that ASS1 activates the mTORC1-SREBP1-SCD5 axis to promote de novo monounsaturated fatty acid synthesis by utilizing acetyl-CoA derived from the glutamine reductive pathway. Treating ASS1-deficient NSCLC cells with erastin combined with arginine deprivation significantly enhanced cell death compared to either treatment alone. Collectively, these results reveal a previously unknown regulatory role of ASS1 in ferroptosis resistance and provide a potential therapeutic target for ASS1-deficient NSCLC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-1999
  6. Proteomics Clin Appl. 2023 Mar 08. e2200106
    Optimized plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial.
    Matthew B O'Rourke, Andrzej S Januszewski, David R Sullivan, Imre Lengyel, Alan J Stewart, Swati Arya, Ronald Ma, Sanjeev Galande, Anandwardhan A Hardikar, Mugdha V Joglekar, Anthony C Keech, Alicia J Jenkins, Mark P Molloy.
      PURPOSE: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow LC-MS analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes.METHODS: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants.
    RESULTS: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard was less than 2%. Fenofibrate altered seven plasma proteins.
    CONCLUSIONS AND CLINICAL RELEVANCE: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balances proteomic depth with time and resource costs. This article is protected by copyright. All rights reserved.
    Keywords:  Plasma; biomarker; diabetes; fenofibrate; mass spectrometry; proteomics
    DOI:  https://doi.org/10.1002/prca.202200106
  7. J Mass Spectrom Adv Clin Lab. 2023 Apr;28 47-55
    Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects.
    S Rischke, L Hahnefeld, B Burla, F Behrens, R Gurke, T J Garrett.
      Mass spectrometry focusing on small endogenous molecules has become an integral part of biomarker discovery in the pursuit of an in-depth understanding of the pathophysiology of various diseases, ultimately enabling the application of personalized medicine. While LC-MS methods allow researchers to gather vast amounts of data from hundreds or thousands of samples, the successful execution of a study as part of clinical research also requires knowledge transfer with clinicians, involvement of data scientists, and interactions with various stakeholders. The initial planning phase of a clinical research project involves specifying the scope and design, and engaging relevant experts from different fields. Enrolling subjects and designing trials rely largely on the overall objective of the study and epidemiological considerations, while proper pre-analytical sample handling has immediate implications on the quality of analytical data. Subsequent LC-MS measurements may be conducted in a targeted, semi-targeted, or non-targeted manner, resulting in datasets of varying size and accuracy. Data processing further enhances the quality of data and is a prerequisite for in-silico analysis. Nowadays, the evaluation of such complex datasets relies on a mix of classical statistics and machine learning applications, in combination with other tools, such as pathway analysis and gene set enrichment. Finally, results must be validated before biomarkers can be used as prognostic or diagnostic decision-making tools. Throughout the study, quality control measures should be employed to enhance the reliability of data and increase confidence in the results. The aim of this graphical review is to provide an overview of the steps to be taken when conducting an LC-MS-based clinical research project to search for small molecule biomarkers.
    Keywords:  (U)HPLC (Ultra-), High pressure liquid chromatography; Biomarker Discovery Study; HILIC, Hydrophilic interaction liquid chromatography; HRMS, High resolution mass spectrometry; LC-MS, Liquid chromatography – mass spectrometry; LC-MS-Based Clinical Research; Lipidomics; MRM, Multiple reaction monitoring; Metabolomics; PCA, Principal component analysis; QA, Quality assurance; QC, Quality control; RF, Random Forest; RP, Reversed phase; SVA, Support vector machine
    DOI:  https://doi.org/10.1016/j.jmsacl.2023.02.003
  8. Cancer Lett. 2023 Mar 07. pii: S0304-3835(23)00070-8. [Epub ahead of print] 216119
    Targeting GPX4 in human cancer: Implications of ferroptosis induction for tackling cancer resilience.
    Jaewang Lee, Jong-Lyel Roh.
      Cancer metabolic alterations have been emphasized to protect cancer cells from cell death. The metabolic reprogramming toward a mesenchymal state makes cancer cells resistant to therapy but vulnerable to ferroptosis induction. Ferroptosis is a new form of regulated cell death based on the iron-dependent accumulation of excessive lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the core regulator of ferroptosis by detoxifying cellular lipid peroxidation using glutathione as a cofactor. GPX4 synthesis requires selenium incorporation into the selenoprotein through isopentenylation and selenocysteine tRNA maturation. GPX4 synthesis and expression can be regulated by multiple levels of its transcription, translation, posttranslational modifications, and epigenetic modifications. Targeting GPX4 in cancer may be a promising strategy for effectively inducing ferroptosis and killing therapy-resistant cancer. Several pharmacological therapeutics targeting GPX4 have been developed constantly to activate ferroptosis induction in cancer. The potential therapeutic index of GPX4 inhibitors remains to be tested with thorough examinations of their safety and adverse effects in vivo and clinical trials. Many papers have been published continuously in recent years, requiring state-of-the-art updates in targeting GPX4 in cancer. Herein, we summarize targeting the GPX4 pathway in human cancer, which leads to implications of ferroptosis induction for tackling cancer resilience.
    Keywords:  Cancer; Ferroptosis; Glutathione peroxidase 4; Lipid peroxidation; Resistance
    DOI:  https://doi.org/10.1016/j.canlet.2023.216119
  9. J Mass Spectrom Adv Clin Lab. 2023 Apr;28 35-46
    Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.
    A Sens, S Rischke, L Hahnefeld, E Dorochow, S M G Schäfer, D Thomas, M Köhm, G Geisslinger, F Behrens, R Gurke.
      The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.
    Keywords:  1-AG, 1-arachidonoyl glycerol; 1-LG, 1-linoleoyl glycerol; 2-AG, 2-arachidonoyl glycerol; 2-LG, 2- linoleoyl glycerol; ACN, acetonitrile; AEA, arachidonoyl ethanolamide; BHT, 2,6-di-tert-butyl-4-methylphenol; CAR, carnitine; EC, endocannabinoid; FC, fold change; FT, freezing temperature/storage in ice water; HETE, hydroxyeicosatetraenoate; HRMS, high-resolution mass spectrometry; IRB, Institutional Review Board; IS, internal standard; K3EDTA plasma sampling; K3EDTA, tripotassium ethylenediaminetetraacetic acid; LC, liquid chromatography; LEA, linoleoyl ethanolamide; LLE, liquid–liquid extraction; LLOQ, lowest limit of quantification; LPA, lysophosphatidic acid; LPC O, lysophosphatidylcholine-ether; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatic inositol; Lipidomics; MS/MS, tandem mass spectrometry; MTBE, methyl tertiary-butyl ether; MeOH, methanol; Metabolomics; OEA, oleoyl ethanolamide; PBS, phosphate-buffered saline; PC, phohsphatidylcholine; PE, phosphotidylethanolamine; PEA, palmitoyl ethanolamide; PI, phosphatidylinositol; Pre-analytics; QC, quality control; REC, Research Ethics Committee; RT, room temperature; Ref, reference sample; SEA, stearoyl ethanolamide; SPE, solid-phase extraction; STD, calibration standard; Sampling protocol; VEA, vaccenic acid ethanolamid; WB, whole blood
    DOI:  https://doi.org/10.1016/j.jmsacl.2023.02.002
  10. Cells. 2023 Mar 04. pii: 804. [Epub ahead of print]12(5):
    Polyunsaturated Fatty Acids Drive Lipid Peroxidation during Ferroptosis.
    Michael S Mortensen, Jimena Ruiz, Jennifer L Watts.
      Ferroptosis is a form of regulated cell death that is intricately linked to cellular metabolism. In the forefront of research on ferroptosis, the peroxidation of polyunsaturated fatty acids has emerged as a key driver of oxidative damage to cellular membranes leading to cell death. Here, we review the involvement of polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs), lipid remodeling enzymes and lipid peroxidation in ferroptosis, highlighting studies revealing how using the multicellular model organism Caenorhabditis elegans contributes to the understanding of the roles of specific lipids and lipid mediators in ferroptosis.
    Keywords:  AA; Caenorhabditis elegans; DGLA; MUFA; PUFA; ferroptosis; lipid peroxidation
    DOI:  https://doi.org/10.3390/cells12050804
  11. Int J Mol Sci. 2023 Feb 26. pii: 4569. [Epub ahead of print]24(5):
    Simple In Vitro 18O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.
    Boris Tupertsev, Sergey Osipenko, Albert Kireev, Eugene Nikolaev, Yury Kostyukevich.
      The identification of drug metabolites formed with different in vitro systems by HPLC-MS is a standard step in preclinical research. In vitro systems allow modeling of real metabolic pathways of a drug candidate. Despite the emergence of various software and databases, identification of compounds is still a complex task. Measurement of the accurate mass, correlation of chromatographic retention times and fragmentation spectra are often insufficient for identification of compounds especially in the absence of reference materials. Metabolites can "slip under the nose", since it is often not possible to reliably confirm that a signal belongs to a metabolite and not to other compounds in complex systems. Isotope labeling has proved to be a tool that aids in small molecule identification. The introduction of heavy isotopes is done with isotope exchange reactions or with complicated synthetic schemes. Here, we present an approach based on the biocatalytic insertion of oxygen-18 isotope under the action of liver microsomes enzymes in the presence of 18O2. Using the local anesthetic bupivacaine as an example, more than 20 previously unknown metabolites were reliably discovered and annotated in the absence of the reference materials. In combination with high-resolution mass spectrometry and modern methods of mass spectrometric metabolism data processing, we demonstrated the ability of the proposed approach to increase the degree of confidence in interpretating metabolism data.
    Keywords:  18O; drug metabolism; mass spectrometry; metabolites identification
    DOI:  https://doi.org/10.3390/ijms24054569
  12. Proteomics. 2023 Mar 06. e2200038
    A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation.
    Jack Penny, Mohammad Arefian, Gunnar N Schroeder, José A Bengoechea, Ben C Collins.
      Data independent acquisition (DIA/SWATH) MS is a primary strategy in quantitative proteomics. diaPASEF is a recent adaptation using trapped ion mobility spectrometry (TIMS) to improve selectivity/sensitivity. Complex DIA spectra are typically analyzed with reference to spectral libraries. The best-established method for generating libraries uses offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation-based libraries. We investigated whether an analogous GPF-based approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data. We developed a rapid library generation approach using an IM-GPF acquisition scheme in the m/z versus 1/K0 space requiring seven injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation. We found that library generation by IM-GPF outperformed direct library generation from diaPASEF and had performance approaching that of the deep library. This establishes the IM-GPF scheme as a pragmatic approach to rapid library generation for analysis of diaPASEF data.
    Keywords:  DIA; SWATH ; diaPASEF; protein complex; protein-protein interaction
    DOI:  https://doi.org/10.1002/pmic.202200038
  13. Elife. 2023 Mar 08. pii: e78654. [Epub ahead of print]12
    Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells.
    Madeleine L Hart, Evan Quon, Anna-Lena B G Vigil, Ian A Engstrom, Oliver J Newsom, Kristian Davidsen, Pia Hoellerbauer, Samantha M Carlisle, Lucas B Sullivan.
      The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC), but mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports human cell proliferation through aspartate synthesis but, unlike other ETC impairments, the effects of SDH inhibition are not ameliorated by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH-impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context depend on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production through pyruvate carboxylation and reductive carboxylation of glutamine. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. These data therefore identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal how compartmentalized redox changes can impact cellular fitness.
    Keywords:  biochemistry; cancer biology; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.78654
  14. Front Oncol. 2023 ;13 1152402
    Editorial: Cancer cell reprogramming: Impact on carcinogenesis and cancer progression.
    Yue Zhao, Simona Kranjc Brezar, Elvira V Grigorieva, Ira-Ida Skvortsova.
      
    Keywords:  cancer metabolism; cancer metastasis; cancer stem cell; carcinogenesis; cell reprogramming
    DOI:  https://doi.org/10.3389/fonc.2023.1152402
  15. Natl Sci Rev. 2023 Jan;10(1): nwac059
    High-throughput site-specific N-glycoproteomics reveals glyco-signatures for liver disease diagnosis.
    Zhenyu Sun, Bin Fu, Guoli Wang, Lei Zhang, Ruofan Xu, Ying Zhang, Haojie Lu.
      The glycoproteome has emerged as a prominent target for screening biomarkers, as altered glycosylation is a hallmark of cancer cells. In this work, we incorporated tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for the multiplexed analysis of intact N-glycopeptides. Benefiting from the complementary nature of two different mass spectrometry dissociation methods for identification and multiplex labeling for quantification of intact N-glycopeptides, we conducted the most comprehensive site-specific and subclass-specific N-glycosylation profiling of human serum immunoglobulin G (IgG) to date. By analysing the serum of 90 human patients with varying severities of liver diseases, as well as healthy controls, we identified that the combination of IgG1-H3N5F1 and IgG4-H4N3 can be used for distinguishing between different stages of liver diseases. Finally, we used targeted parallel reaction monitoring to successfully validate the expression changes of glycosylation in liver diseases in a different sample cohort that included 45 serum samples.
    Keywords:  N-glycopeptides; glycoproteomics; high-throughput; liver disease; quantification
    DOI:  https://doi.org/10.1093/nsr/nwac059
  16. Talanta. 2023 Mar 04. pii: S0039-9140(23)00167-4. [Epub ahead of print]258 124416
    Sequential isolation of metabolites and lipids from a single sample to achieve multiomics by using TRIzol reagent.
    Cheng-Yen Kao, Chung-Te Chang, Pei-Yun Kuo, Chia-Jen Lin, Huai-Hsuan Chiu, Hsiao-Wei Liao.
      Simultaneous extraction of various types of biomolecule from a single sample can be beneficial for multiomics studies of unique specimens. An efficient and convenient sample preparation approach must be developed that can comprehensively isolate and extract biomolecules from one sample. TRIzol reagent is widely used in biological studies for DNA, RNA, and protein isolation. This study evaluated the feasibility of using TRIzol reagent for the simultaneous isolation of not only DNA, RNA, and proteins but also metabolites and lipids from a single sample. Through the comparison of known metabolites and lipids obtained using the conventional methanol (MeOH) and methyl-tert-butyl ether (MTBE) extraction methods, we determined the presence of metabolites and lipids in the supernatant during TRIzol sequential isolation. Finally, we performed untargeted metabolomics and lipidomics to examine metabolite and lipid alterations associated with the jhp0417 mutation in Helicobacter pylori by using the TRIzol sequential isolation protocol and MeOH and MTBE extraction methods. Metabolites and lipids with significant differences isolated using the TRIzol sequential isolation protocol were consistent with those obtained using the conventional MeOH and MTBE extraction methods. These results indicated that TRIzol reagent can be used to simultaneously isolate metabolites and lipids from a single sample. Thus, TRIzol reagent can be used in biological and clinical research, especially in multiomics studies.
    Keywords:  Lipidomics; Metabolomics; Multiomics; Sequential isolation; TRIzol
    DOI:  https://doi.org/10.1016/j.talanta.2023.124416
  17. Pulm Circ. 2023 Jan;13(1): e12205
    Transpulmonary amino acid metabolism in the sugen hypoxia model of pulmonary hypertension.
    Nicolas Philip, Hongyang Pi, Mahin Gadkari, Xin Yun, John Huetsch, Cissy Zhang, Robert Harlan, Aurelie Roux, David Graham, Larissa Shimoda, Anne Le, Scott Visovatti, Peter J Leary, Sina A Gharib, Catherine Simpson, Lakshmi Santhanam, Jochen Steppan, Karthik Suresh.
      In pulmonary artery hypertension (PAH), emerging evidence suggests that metabolic abnormalities may be contributing to cellular dysfunction in PAH. Metabolic abnormalities such as glycolytic shift have been observed intracellularly in several cell types in PAH, including microvacular endothelial cells (MVECs). Concurrently, metabolomics of human PAH samples has also revealed a variety of metabolic abnormalities; however the relationship between the intracellular metabolic abnormalities and the serum metabolome in PAH remains under investigation. In this study, we utilize the sugen/hypoxia (SuHx) rodent model of PAH to examine the RV, LV and MVEC intracellular metabolome (using targeted metabolomics) in normoxic and SuHx rats. We additionally validate key findings from our metabolomics experiments with data obtained from cell culture of normoxic and SuHx MVECs, as well as metabolomics of human serum samples from two different PAH patient cohorts. Taken together, our data, spanning rat serum, human serum and primary isolated rat MVECs reveal that: (1) key classes of amino acids (specifically, branched chain amino acids-BCAA) are lower in the pre-capillary (i.e., RV) serum of SuHx rats (and humans); (2) intracellular amino acid levels (in particular BCAAs) are increased in SuHx-MVECs; (3) there may be secretion rather than utilization of amino acids across the pulmonary microvasculature in PAH and (4) an oxidized glutathione gradient is present across the pulmonary vasculature, suggesting a novel fate for increased glutamine uptake (i.e., as a source of glutathione). in MVECs in PAH. In summary, these data reveal new insight into the shifts in amino acid metabolism occurring across the pulmonary circulation in PAH.
    Keywords:  PAH; amino acid metabolism; endothelial cells; metabolomics
    DOI:  https://doi.org/10.1002/pul2.12205
  18. Lab Invest. 2023 Jan 10. pii: S0023-6837(22)03990-3. [Epub ahead of print]103(4): 100052
    Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.
    Bradley L Ackermann, Ryan D Morrison, Salisha Hill, Matthew D Westfall, Brent D Butts, Michael D Soper, Jeff A Fill, Andrew E Schade, Daniel C Liebler, Aaron M Gruver.
      Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-μm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted β-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.
    Keywords:  formalin-fixed tissue; mass spectrometry; quantitative proteomics; single section
    DOI:  https://doi.org/10.1016/j.labinv.2022.100052
  19. Chembiochem. 2023 Mar 07. e202300082
    Bioorthogonal Peptide Enrichment From Complex Samples Using a Rink-Amide-Based Catch-and-Release Strategy.
    Tyrza van Leeuwen, Ward Doelman, Robin W R van den Kieboom, Bogdan I Florea, Sander Izaak van Kasteren.
      Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics - the ideal analysis tool in this case - requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2+3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures.
    Keywords:  click-antigens antigen processing chemical proteomics bioorthogonal chemistry mass spectrometry
    DOI:  https://doi.org/10.1002/cbic.202300082
  20. Biochemistry. 2023 Mar 07.
    Lipidome of the Bacteroides Genus Containing New Peptidolipid and Sphingolipid Families Revealed by Multiple-Stage Mass Spectrometry.
    Cheryl F Frankfater, Mariana G Sartorio, Ezequiel Valguarnera, Mario F Feldman, Fong-Fu Hsu.
      The anaerobic bacteria of the Bacteroides fragilis group including Bacteroides thetaiotaomicron, B. fragilis, Bacteroides vulgatus, and Bacteroides ovatus in genus Bacteroides are among the most commonly found human gut microbiota. They are generally commensal but are also opportunistic pathogens. Both the inner and outer membranes of the Bacteroides cell envelope contain abundant lipids with diversified structures, and dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we describe mass spectrometry-based approaches to delineate in detail the lipidome of the membrane and the outer membrane vesicle of the bacteria cells. We identified 15 lipid class/subclasses (>100 molecular species), including sphingolipid families [dihydroceramide (DHC), glycylseryl (GS) DHC, DHC-phosphoinositolphosphoryl-DHC (DHC-PIP-DHC), ethanolamine phosphorylceramide, inositol phosphorylceramide (IPC), serine phosphorylceramide, ceramide-1-phosphate, and glycosyl ceramide], phospholipids [phosphatidylethanolamine, phosphatidylinositol (PI), and phosphatidylserine], peptide lipids (GS-, S-, and G-lipids) and cholesterol sulfate, of which several have not been reported previously, or have similar structures to those found in Porphyromonas gingivalis, the periodontopathic bacterium in oral microbiota. The new DHC-PIPs-DHC lipid family is found only in B. vulgatus, which, however, lacks the PI lipid family. The galactosyl ceramide family is exclusively present in B. fragilis, which nevertheless lacks IPC and PI lipids. The lipidomes as revealed in this study demonstrate the lipid diversity among the various strains and the utility of multiple-stage mass spectrometry (MSn) with high-resolution mass spectrometry in the structural elucidation of complex lipids.
    DOI:  https://doi.org/10.1021/acs.biochem.2c00664
  21. Front Oncol. 2023 ;13 1144821
    Editorial: Targeting cancer cell intracellular metabolism as a strategy against therapy resistance.
    Lingling Liu, Xiaohong Cai, Yuan Zhou, Chengsi He, Hongsheng Li, Xiangfu Liu, Zhengzhi Zou.
      
    Keywords:  cancer metabolism; cancer treatment; drug resistance; immune infiltration; tumor prognosis
    DOI:  https://doi.org/10.3389/fonc.2023.1144821
  22. Methods Mol Biol. 2023 ;2618 219-237
    Characterization of Dendritic Cell Metabolism by Flow Cytometry.
    Eline C Brombacher, Thiago A Patente, Marjolein Quik, Bart Everts.
      In response to different stimuli, dendritic cells (DCs) undergo metabolic reprogramming to support their function. Here we describe how fluorescent dyes and antibody-based approaches can be used to assess various metabolic parameters of DCs including glycolysis, lipid metabolism, mitochondrial activity, and the activity of important sensors and regulators of cellular metabolism, mTOR and AMPK. These assays can be performed using standard flow cytometry and will allow for the determination of metabolic properties of DC populations at single-cell level and to characterize metabolic heterogeneity within them.
    Keywords:  Dendritic cells; Flow cytometry; Glucose; Lipids; Metabolism; Mitochondria; ROS
    DOI:  https://doi.org/10.1007/978-1-0716-2938-3_16
  23. Front Nutr. 2023 ;10 1113739
    Lactate in the tumor microenvironment: A rising star for targeted tumor therapy.
    Zhangzuo Li, Qi Wang, Xufeng Huang, Mengting Yang, Shujing Zhou, Zhengrui Li, Zhengzou Fang, Yidan Tang, Qian Chen, Hanjin Hou, Li Li, Fei Fei, Qiaowei Wang, Yuqing Wu, Aihua Gong.
      Metabolic reprogramming is one of fourteen hallmarks of tumor cells, among which aerobic glycolysis, often known as the "Warburg effect," is essential to the fast proliferation and aggressive metastasis of tumor cells. Lactate, on the other hand, as a ubiquitous molecule in the tumor microenvironment (TME), is generated primarily by tumor cells undergoing glycolysis. To prevent intracellular acidification, malignant cells often remove lactate along with H+, yet the acidification of TME is inevitable. Not only does the highly concentrated lactate within the TME serve as a substrate to supply energy to the malignant cells, but it also works as a signal to activate multiple pathways that enhance tumor metastasis and invasion, intratumoral angiogenesis, as well as immune escape. In this review, we aim to discuss the latest findings on lactate metabolism in tumor cells, particularly the capacity of extracellular lactate to influence cells in the tumor microenvironment. In addition, we examine current treatment techniques employing existing medications that target and interfere with lactate generation and transport in cancer therapy. New research shows that targeting lactate metabolism, lactate-regulated cells, and lactate action pathways are viable cancer therapy strategies.
    Keywords:  immune cells; immunity; lactate; metabolic; tumor microenvironment
    DOI:  https://doi.org/10.3389/fnut.2023.1113739
  24. Nat Methods. 2023 Mar;20(3): 327-330
    Mass spectrometry imaging: the rise of spatially resolved single-cell omics.
    Hua Zhang, Daniel G Delafield, Lingjun Li.
      
    DOI:  https://doi.org/10.1038/s41592-023-01774-6
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