bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021–01–24
forty papers selected by
Giovanny Rodríguez Blanco, University of Edinburgh
  1. The metabolism of cancer cells during metastasis.
  2. Beyond Proteostasis: Lipid Metabolism as a New Player in ER Homeostasis.
  3. Tumor Metabolome: Therapeutic Opportunities Targeting Cancer Metabolic Reprogramming.
  4. Linking Gut Microbiome and Lipid Metabolism: Moving beyond Associations.
  5. Mass spectrometry imaging of phosphatidylcholine metabolism in lungs administered with therapeutic surfactants and isotopic tracers.
  6. Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.
  7. Mass-spectrometric investigations of caloric restriction mimetics.
  8. High-defined quantitative snapshots of the ganglioside lipidome using high resolution ion mobility SLIM assisted shotgun lipidomics.
  9. High Throughput Semiquantitative UHPSFC-MS/MS Lipid Profiling and Lipid Class Determination.
  10. Quantification of All-Trans Retinoic Acid by Liquid Chromatography-Tandem Mass Spectrometry and Association with Lipid Profile in Patients with Type 2 Diabetes.
  11. Exogenous Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells.
  12. Urinary proteome profiling for stratifying patients with familial Parkinson's disease.
  13. Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity.
  14. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures.
  15. Membrane Lipids and Transporter Function.
  16. Cognitive analysis of metabolomics data for systems biology.
  17. Simultaneous Measurement of Amino Acid Enantiomers in Aged Mouse Brain Samples by LC/MS/MS Combined with Derivatization Using Nα-(5-Fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA).
  18. A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.
  19. Serum lipoprotein-derived fatty acids regulate hypoxia-inducible factor.
  20. Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase.
  21. L-Carnitine and Acylcarnitines: Mitochondrial Biomarkers for Precision Medicine.
  22. DaDIA: Hybridizing Data-Dependent and Data-Independent Acquisition Modes for Generating High-Quality Metabolomic Data.
  23. A perspective on the standards describing mass spectrometry-based metabolic phenotyping (metabolomics/metabonomics) studies in publications.
  24. Human Plasma Metabolomics for Biomarker Discovery: Targeting the Molecular Subtypes in Breast Cancer.
  25. Advances in quantitative high-throughput phosphoproteomics with sample multiplexing.
  26. Imaging and analysis of isomeric unsaturated lipids through online photochemical derivatization of C=C bonds.
  27. Ethical Principles, Constraints and Opportunities in Clinical Proteomics.
  28. An Ion Chromatography-Ultrahigh-Resolution-MS1/Data-Independent High-Resolution MS2 Method for Stable Isotope-Resolved Metabolomics Reconstruction of Central Metabolic Networks.
  29. ABHD5 suppresses cancer cell anabolism through lipolysis-dependent activation of the AMPK/mTORC1 pathway.
  30. Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.
  31. Exploring Phosphoinositide Binding Using Native Mass Spectrometry.
  32. Glioma cells require one-carbon metabolism to survive glutamine starvation.
  33. Metabolic consequences for mice lacking Endosialin: LC-MS/MS-based metabolic phenotyping of serum from C56Bl/6J Control and CD248 knock-out mice.
  34. Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.
  35. Nicotinamide N-methyltransferase: at the crossroads between cellular metabolism and epigenetic regulation.
  36. Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer.
  37. Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.
  38. Pharmacological activation of pyruvate kinase M2 reprograms glycolysis leading to TXNIP depletion and AMPK activation in breast cancer cells.
  39. Application of comprehensive lipidomics to biomarker research on adverse drug reactions.
  40. A functional role for eicosanoid-lysophospholipids in activating monocyte signaling.
  1. Nat Rev Cancer. 2021 Jan 18.
    The metabolism of cancer cells during metastasis.
    Gabriele Bergers, Sarah-Maria Fendt.
      Metastasis formation is the major cause of death in most patients with cancer. Despite extensive research, targeting metastatic seeding and colonization is still an unresolved challenge. Only recently, attention has been drawn to the fact that metastasizing cancer cells selectively and dynamically adapt their metabolism at every step during the metastatic cascade. Moreover, many metastases display different metabolic traits compared with the tumours from which they originate, enabling survival and growth in the new environment. Consequently, the stage-dependent metabolic traits may provide therapeutic windows for preventing or reducing metastasis, and targeting the new metabolic traits arising in established metastases may allow their eradication.
    DOI:  https://doi.org/10.1038/s41568-020-00320-2
  2. Metabolites. 2021 Jan 14. pii: E52. [Epub ahead of print]11(1):
    Beyond Proteostasis: Lipid Metabolism as a New Player in ER Homeostasis.
    Jiaming Xu, Stefan Taubert.
      Biological membranes are not only essential barriers that separate cellular and subcellular structures, but also perform other critical functions such as the initiation and propagation of intra- and intercellular signals. Each membrane-delineated organelle has a tightly regulated and custom-made membrane lipid composition that is critical for its normal function. The endoplasmic reticulum (ER) consists of a dynamic membrane network that is required for the synthesis and modification of proteins and lipids. The accumulation of unfolded proteins in the ER lumen activates an adaptive stress response known as the unfolded protein response (UPR-ER). Interestingly, recent findings show that lipid perturbation is also a direct activator of the UPR-ER, independent of protein misfolding. Here, we review proteostasis-independent UPR-ER activation in the genetically tractable model organism Caenorhabditis elegans. We review the current knowledge on the membrane lipid composition of the ER, its impact on organelle function and UPR-ER activation, and its potential role in human metabolic diseases. Further, we summarize the bi-directional interplay between lipid metabolism and the UPR-ER. We discuss recent progress identifying the different respective mechanisms by which disturbed proteostasis and lipid bilayer stress activate the UPR-ER. Finally, we consider how genetic and metabolic disturbances may disrupt ER homeostasis and activate the UPR and discuss how using -omics-type analyses will lead to more comprehensive insights into these processes.
    Keywords:  endoplasmic reticulum; lipid bilayer stress; lipidomics; phosphatidylcholine; unfolded protein response; unsaturated fatty acid
    DOI:  https://doi.org/10.3390/metabo11010052
  3. Cancers (Basel). 2021 Jan 16. pii: E314. [Epub ahead of print]13(2):
    Tumor Metabolome: Therapeutic Opportunities Targeting Cancer Metabolic Reprogramming.
    Javier Márquez, José M Matés.
      The study of cancer metabolism is regaining center stage and becoming a hot topic in tumor biology and clinical research, after a period where such kind of experimental approaches were somehow forgotten or disregarded in favor of powerful functional genomic and proteomic studies [...].
    DOI:  https://doi.org/10.3390/cancers13020314
  4. Metabolites. 2021 Jan 15. pii: E55. [Epub ahead of print]11(1):
    Linking Gut Microbiome and Lipid Metabolism: Moving beyond Associations.
    Santosh Lamichhane, Partho Sen, Marina Amaral Alves, Henrique C Ribeiro, Peppi Raunioniemi, Tuulia Hyötyläinen, Matej Orešič.
      Various studies aiming to elucidate the role of the gut microbiome-metabolome co-axis in health and disease have primarily focused on water-soluble polar metabolites, whilst non-polar microbial lipids have received less attention. The concept of microbiota-dependent lipid biotransformation is over a century old. However, only recently, several studies have shown how microbial lipids alter intestinal and circulating lipid concentrations in the host, thus impacting human lipid homeostasis. There is emerging evidence that gut microbial communities play a particularly significant role in the regulation of host cholesterol and sphingolipid homeostasis. Here, we review and discuss recent research focusing on microbe-host-lipid co-metabolism. We also discuss the interplay of human gut microbiota and molecular lipids entering host systemic circulation, and its role in health and disease.
    Keywords:  gut; lipidomics; lipids; metabolomics; microbiome
    DOI:  https://doi.org/10.3390/metabo11010055
  5. J Lipid Res. 2021 Jan 13. pii: S0022-2275(21)00003-1. [Epub ahead of print] 100023
    Mass spectrometry imaging of phosphatidylcholine metabolism in lungs administered with therapeutic surfactants and isotopic tracers.
    Shane R Ellis, Emily Hall, Madhuriben Panchal, Bryn Flinders, Jens Madsen, Grielof Koster, Ron M A Heeren, Howard W Clark, Anthony D Postle.
      Mass spectrometry imaging (MSI) visualises molecular distributions throughout tissues but is blind to dynamic metabolic processes. Here, MSI with high mass resolution together with multiple stable isotope labelling provided spatial analyses of phosphatidylcholine (PC) metabolism in mouse lungs. Dysregulated surfactant metabolism is central to many respiratory diseases. Metabolism and turnover of therapeutic pulmonary surfactants were imaged from distributions of intact and metabolic products of an added tracer, universally 13C-labelled dipalmitoyl PC (U13C-DPPC). The parenchymal distributions of newly synthesised PC species were also imaged from incorporations of methyl-D9-choline. This dual labelling strategy demonstrated both lack of inhibition of endogenous PC synthesis by exogenous surfactant and location of acyl chain remodelling processes acting on the U13C-DPPC-labelled surfactant, leading to formation of polyunsaturated PC lipids. This ability to visualise discrete metabolic events will greatly enhance our understanding of lipid metabolism in diverse tissues, and has potential application to both clinical and experimental studies.
    Keywords:  MALDI; Pulmonary surfactant; isotope labelling; lipids; mass spectrometry imaging; metabolism; phosphatidylcholine; remodeling
    DOI:  https://doi.org/10.1016/j.jlr.2021.100023
  6. Methods Mol Biol. 2021 ;2251 39-53
    Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.
    Gaëtan Chicanne, Justine Bertrand-Michel, Julien Viaud, Karim Hnia, Jonathan Clark, Bernard Payrastre.
      Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.
    Keywords:  Fatty acyl chains; LC-MS; Molecular species; Phosphoinositides; Platelets
    DOI:  https://doi.org/10.1007/978-1-0716-1142-5_3
  7. Proteomics. 2021 Jan 18. e2000121
    Mass-spectrometric investigations of caloric restriction mimetics.
    Michael J Bibyk, Melanie J Campbell, Amanda B Hummon.
      Caloric Restriction (CR) is an innovative therapy used in tumor tissue and tumor model studies to promote cell death and decrease cell viability. Caloric restriction mimetics (CRM) are a class of drugs that induce CR and starvation conditions within a cell. When used simultaneously with other chemotherapy agents, the effects are synergistic and effective at promoting tumor cell death. In this review, we discuss CRMs and their potential as cancer therapeutics. Firstly, we establish an overview of CR and its impacts on healthy and tumor cells. CR and CRM drugs have shown to decrease age-related diseases and can act as an anti-cancer agent. As it can be challenging for an individual to diligently stick to a diet that would induce CR, CRMs are even more desirable. Then we discuss the drug class by highlighting three CRMs; resveratrol, (-)-hydroxycitric acid and rapamycin. These CRMs are commonly known for their dietary effects, but the underlying mechanisms that drive cellular metabolic and proteomic changes show promise as a cancer therapeutic. Lastly, we highlight the use of mass spectrometry and proteomic techniques on experiments utilizing CRM drugs to understand the cellular pathways impacted by this drug class, leading to a better understanding of the anti-cancer properties and potentials of caloric restriction mimetics. This article is protected by copyright. All rights reserved.
    Keywords:  Anti-cancer; caloric restriction; caloric restriction mimetic; hydroxycitric acid; mass spectrometry; proteomics; rapamycin; resveratrol
    DOI:  https://doi.org/10.1002/pmic.202000121
  8. Anal Chim Acta. 2021 Feb 15. pii: S0003-2670(20)31215-0. [Epub ahead of print]1146 77-87
    High-defined quantitative snapshots of the ganglioside lipidome using high resolution ion mobility SLIM assisted shotgun lipidomics.
    Kelly L Wormwood Moser, Gregory Van Aken, Daniel DeBord, Nathan Galen Hatcher, Laura Maxon, Melissa Sherman, Lihang Yao, Kim Ekroos.
      Defects in sphingolipid metabolism have emerged as a common link across neurodegenerative disorders, and a deeper understanding of the lipid content in preclinical models and patient specimens offers opportunities for development of new therapeutic targets and biomarkers. Sphingolipid metabolic pathways include the formation of glycosphingolipid species that branch into staggeringly complex structural heterogeneity within the globoside and ganglioside sub-lipidomes. Characterization of these sub-lipidomes has typically relied on liquid chromatography-mass spectrometry-based (LC-MS) approaches, but such assays are challenging and resource intensive due to the close structural heterogeneity, the presence of isobaric and isomeric species, and broad dynamic range of endogenous glycosphingolipids. Here, we apply Structures for Lossless Ion Manipulations (SLIM)-based High Resolution Ion Mobility (HRIM)-MS to enable rapid, repeatable, quantitative assays with deep structural information sufficient to resolve endogenous brain gangliosides at the level of individual molecular species. Analyses were performed using a prototype SLIM-MS instrument equipped with a 13-m serpentine path which enabled resolution of closely related isomeric analytes such as GD1a d36:1 and GD1b d36:1 based on recorded mass-to-charge (m/z) and arrival times. To demonstrate the power of our methodology, brain extracts derived from wild-type mice hemi-brains were analyzed by HRIM-MS using flow injection analyses (FIA) without the need for additional separation by liquid chromatography. Endogenous ganglioside species were readily resolved, identified, and quantified by FIA-SLIM-MS analyses within 2 min per sample. Thus, the FIA-SLIM-MS platform enables robust quantification across a broad range of lipid species in biological specimens in a standardized assay format that is readily scalable to support studies with large sample numbers.
    Keywords:  Gangliosides; High resolution ion mobility; Isomers; Lipidomics; Mass spectrometry
    DOI:  https://doi.org/10.1016/j.aca.2020.12.022
  9. J Chromatogr Sci. 2021 Jan 22. pii: bmaa121. [Epub ahead of print]
    High Throughput Semiquantitative UHPSFC-MS/MS Lipid Profiling and Lipid Class Determination.
    Zdenka Bartosova, Susana Villa Gonzalez, André Voigt, Per Bruheim.
      High throughput and high-resolution lipid analyses are important for many biological model systems and research questions. This comprises both monitoring at the individual lipid species level and broad lipid classes. Here, we present a nontarget semiquantitative lipidomics workflow based on ultrahigh performance supercritical fluid chromatography (UHPSFC)-mass spectrometry (MS). The optimized chromatographic conditions enable the base-line separation of both nonpolar and polar classes in a single 7-minute run. Ionization efficiencies of lipid classes vary 10folds in magnitude and great care must be taken in a direct interpretation of raw data. Therefore, the inclusion of internal standards or experimentally determined Response factors (RF) are highly recommended for the conversion of raw abundances into (semi) quantitative data. We have deliberately developed an algorithm for automatic semiquantification of lipid classes by RF. The workflow was tested and validated using a bovine liver extract with satisfactory results. The RF corrected data provide a more representative relative lipid class determination, but also the interpretation of individual lipid species should be performed on RF corrected data. In addition, semiquantification can be improved by using internal or also external standards when more accurate quantitative data are of interest but this requires validation for all new sample types. The workflow established greatly extends the potential of nontarget UHPSFC-MS/MS based analysis.
    DOI:  https://doi.org/10.1093/chromsci/bmaa121
  10. Metabolites. 2021 Jan 19. pii: E60. [Epub ahead of print]11(1):
    Quantification of All-Trans Retinoic Acid by Liquid Chromatography-Tandem Mass Spectrometry and Association with Lipid Profile in Patients with Type 2 Diabetes.
    Jakob Morgenstern, Thomas Fleming, Elisabeth Kliemank, Maik Brune, Peter Nawroth, Andreas Fischer.
      Retinoic acids are vitamin A metabolites that have numerous essential functions in humans, and are also used as drugs to treat acne and acute promyelocytic leukemia. All-trans retinoic acid (atRA) is the major occurring metabolite of retinoic acid in humans. This study provides a sensitive and specific liquid chromatography-tandem mass spectrometry approach in order to quantify atRA in human plasma samples. The isolation of atRA by hyperacidified liquid-liquid extraction using hexane and ethyl acetate resulted in a recovery of 89.7 ± 9.2%. The lower limit of detection was 20 pg·mL-1, and 7 point calibration displayed good linearity (R2 = 0.994) in the range of 50-3200 pg mL-1. Selectivity was guaranteed by the use of two individual mass transitions (qualifier and quantifier), and precision and accuracy were determined intraday and interday with a coefficient variation of 9.3% (intraday) and 14.0% (interday). Moreover, the method could be used to isolate atRA from hyperlipidemic samples. Applying this method to plasma samples from patients with poorly controlled Type 2 diabetes significantly decreased atRA plasma levels as compared to those of the healthy controls. In addition, atRA concentrations were highly associated with increased low-density lipoprotein (LDL) and decreased high-density lipoprotein (HDL) cholesterol levels.
    Keywords:  hyperlipidemia; liquid chromatography–tandem mass spectrometry; liquid–liquid extraction; retinoic acid
    DOI:  https://doi.org/10.3390/metabo11010060
  11. Cells. 2021 Jan 16. pii: E175. [Epub ahead of print]10(1):
    Exogenous Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells.
    Angela Maria Rizzo, Irma Colombo, Gigliola Montorfano, Stefania Zava, Paola Antonia Corsetto.
      (1) Background: Lipid metabolism is a fundamental hallmark of all tumors, especially of breast cancer. Few studies describe the different lipid metabolisms and sensitivities to the microenvironment of breast cancer cell subtypes that influence the proliferation, aggressiveness, and success of therapy. This study describes the impact of lipid microenvironment on endoplasmic reticulum (ER) membrane and metabolic activity in two breast cancer cell lines with Luminal A and triple-negative breast cancer (TNBC) features. (2) Methods: We investigated the peculiar lipid phenotype of a TNBC cell line, MDA-MB-231, and a Luminal A cell line, MCF7, and their different sensitivity to exogenous fatty acids (i.e., palmitic acid (PA) and docosahexaenoic acid (DHA)). Moreover, we verified the impact of exogenous fatty acids on ER lipid composition. (3) Results: The data obtained demonstrate that MDA-MB-231 cells are more sensitive to the lipid microenvironment and that both PA and DHA are able to remodel their ER membranes with consequences on resident enzyme activity. On the contrary, MCF7 cells are less sensitive to PA, whereas they incorporate DHA, although less efficiently than MDA-MB-231 cells. (4) Conclusions: This study sustains the importance of lipid metabolism as an innovative hallmark to discriminate breast cancer subclasses and to develop personalized and innovative pharmacological strategies. The different sensitivities to the lipid environment shown by MCF7 and MDA-MB-231 cells might be related to cell malignancy and chemoresistance onset. In the future, this new approach could lead to a substantial decrease both in deleterious side effects for the patients and in the cost of entire therapeutic treatments coupled with increased therapy efficiency.
    Keywords:  ER; cancer; fatty acids; lipid metabolism
    DOI:  https://doi.org/10.3390/cells10010175
  12. EMBO Mol Med. 2021 Jan 22. e13257
    Urinary proteome profiling for stratifying patients with familial Parkinson's disease.
    Sebastian Virreira Winter, Ozge Karayel, Maximilian T Strauss, Shalini Padmanabhan, Matthew Surface, Kalpana Merchant, Roy N Alcalay, Matthias Mann.
      The prevalence of Parkinson's disease (PD) is increasing but the development of novel treatment strategies and therapeutics altering the course of the disease would benefit from specific, sensitive, and non-invasive biomarkers to detect PD early. Here, we describe a scalable and sensitive mass spectrometry (MS)-based proteomic workflow for urinary proteome profiling. Our workflow enabled the reproducible quantification of more than 2,000 proteins in more than 200 urine samples using minimal volumes from two independent patient cohorts. The urinary proteome was significantly different between PD patients and healthy controls, as well as between LRRK2 G2019S carriers and non-carriers in both cohorts. Interestingly, our data revealed lysosomal dysregulation in individuals with the LRRK2 G2019S mutation. When combined with machine learning, the urinary proteome data alone were sufficient to classify mutation status and disease manifestation in mutation carriers remarkably well, identifying VGF, ENPEP, and other PD-associated proteins as the most discriminating features. Taken together, our results validate urinary proteomics as a valuable strategy for biomarker discovery and patient stratification in PD.
    Keywords:  DIA; Parkinson’s disease; biomarker; mass spectrometry; urinary proteome
    DOI:  https://doi.org/10.15252/emmm.202013257
  13. J Clin Invest. 2021 01 19. pii: 136055. [Epub ahead of print]131(2):
    Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity.
    Rohit Sharma, Bryn Reinstadler, Kristin Engelstad, Owen S Skinner, Erin Stackowitz, Ronald G Haller, Clary B Clish, Kerry Pierce, Melissa A Walker, Robert Fryer, Devin Oglesbee, Xiangling Mao, Dikoma C Shungu, Ashok Khatri, Michio Hirano, Darryl C De Vivo, Vamsi K Mootha.
      Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.
    Keywords:  Genetics; Intermediary metabolism; Metabolism; Mitochondria; Monogenic diseases; RET; HS6ST1; sE-selectin; integrated stress response; creatine; pyruvate; 2-hydroxybutyrate; alpha-hydroxybutyrate; lactoyl-amino acids; hydroxy-fatty acids; hydroxy-acylcarnitines
    DOI:  https://doi.org/10.1172/JCI136055
  14. J Lipid Res. 2020 Dec;pii: S0022-2275(20)60017-7. [Epub ahead of print]61(12): 1539-1555
    Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures.
    Gerhard Liebisch, Eoin Fahy, Junken Aoki, Edward A Dennis, Thierry Durand, Christer S Ejsing, Maria Fedorova, Ivo Feussner, William J Griffiths, Harald Köfeler, Alfred H Merrill, Robert C Murphy, Valerie B O'Donnell, Olga Oskolkova, Shankar Subramaniam, Michael J O Wakelam, Friedrich Spener.
      A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.
    Keywords:  fatty acyls; glycerolipids; glycerophospholipids; lipidomics; mass spectrometry; sphingolipids; sterols
    DOI:  https://doi.org/10.1194/jlr.S120001025
  15. Biochim Biophys Acta Mol Basis Dis. 2021 Jan 18. pii: S0925-4439(21)00012-0. [Epub ahead of print] 166079
    Membrane Lipids and Transporter Function.
    Bruno Stieger, Julia Steiger, Kaspar P Locher.
      Transport proteins are essential for cells in allowing the exchange of substances between cells and their environment across the lipid bilayer forming a tight barrier. Membrane lipids modulate the function of transmembrane proteins such as transporters in two ways: Lipids are tightly and specifically bound to transport proteins and in addition they modulate from the bulk of the lipid bilayer the function of transport proteins. This overview summarizes currently available information at the ultrastructural level on lipids tightly bound to transport proteins and the impact of altered bulk membrane lipid composition. Human diseases leading to altered lipid homeostasis will lead to altered membrane lipid composition, which in turn affect the function of transporter proteins.
    Keywords:  human lipid homeostasis; lipids; plasma membrane composition; transport protein
    DOI:  https://doi.org/10.1016/j.bbadis.2021.166079
  16. Nat Protoc. 2021 Jan 22.
    Cognitive analysis of metabolomics data for systems biology.
    Erica L-W Majumder, Elizabeth M Billings, H Paul Benton, Richard L Martin, Amelia Palermo, Carlos Guijas, Markus M Rinschen, Xavier Domingo-Almenara, J Rafael Montenegro-Burke, Bradley A Tagtow, Robert S Plumb, Gary Siuzdak.
      Cognitive computing is revolutionizing the way big data are processed and integrated, with artificial intelligence (AI) natural language processing (NLP) platforms helping researchers to efficiently search and digest the vast scientific literature. Most available platforms have been developed for biomedical researchers, but new NLP tools are emerging for biologists in other fields and an important example is metabolomics. NLP provides literature-based contextualization of metabolic features that decreases the time and expert-level subject knowledge required during the prioritization, identification and interpretation steps in the metabolomics data analysis pipeline. Here, we describe and demonstrate four workflows that combine metabolomics data with NLP-based literature searches of scientific databases to aid in the analysis of metabolomics data and their biological interpretation. The four procedures can be used in isolation or consecutively, depending on the research questions. The first, used for initial metabolite annotation and prioritization, creates a list of metabolites that would be interesting for follow-up. The second workflow finds literature evidence of the activity of metabolites and metabolic pathways in governing the biological condition on a systems biology level. The third is used to identify candidate biomarkers, and the fourth looks for metabolic conditions or drug-repurposing targets that the two diseases have in common. The protocol can take 1-4 h or more to complete, depending on the processing time of the various software used.
    DOI:  https://doi.org/10.1038/s41596-020-00455-4
  17. Metabolites. 2021 Jan 15. pii: E57. [Epub ahead of print]11(1):
    Simultaneous Measurement of Amino Acid Enantiomers in Aged Mouse Brain Samples by LC/MS/MS Combined with Derivatization Using Nα-(5-Fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA).
    Taiji Yamamoto, Keisuke Yaku, Takashi Nakagawa.
      D-amino acids have distinct roles from their l-enantiomer. In particular, some D-amino acids function as agonists or antagonists of neuronal receptors and are involved in higher brain functions. Thus, it is important to precisely measure the levels of these amino acid enantiomers in cells and tissues. Various quantification methods have been developed for measurements of chiral amino acids. However, each method has advantages and disadvantages. Additionally, measuring the amino acid enantiomers in crude biological samples requires a higher selectivity. In this study, we developed a quantification method for amino acid enantiomers using derivatization with Nα-(5-Fluoro-2,4-dinitrophenyl)-l-leucinamide (l-FDLA) followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a conventional reversed-phase column. We simultaneously identified 10 chiral amino acids. Furthermore, we applied this method to investigate murine tissue samples and examined the effect of aging on the amino acid levels in aged brain regions. We found that aging decreased the levels of both D-serine and D-aspartate in the hippocampus. In addition, D-Phenylalanine in the thalamus significantly increased with age. In conclusion, our method is suitable for the quantification of the D-amino acids in crude biological samples and may contribute to elucidating the biological roles of chiral amino acids.
    Keywords:  D-amino acid; LC/MS/MS; aging; brain; l-FDLA
    DOI:  https://doi.org/10.3390/metabo11010057
  18. Anal Bioanal Chem. 2021 Jan 18.
    A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.
    Lisa Kobos, Christina R Ferreira, Tiago J P Sobreira, Bartek Rajwa, Jonathan Shannahan.
      Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.
    Keywords:  Animal models; Lipid signaling; Lipid storage; Lipidomics; Phosphatidylethanolamines; Pre-analytical methods
    DOI:  https://doi.org/10.1007/s00216-020-03151-0
  19. J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50699-7. [Epub ahead of print]295(52): 18284-18300
    Serum lipoprotein-derived fatty acids regulate hypoxia-inducible factor.
    Wei Shao, Jiwon Hwang, Chune Liu, Debaditya Mukhopadhyay, Shan Zhao, Meng-Chieh Shen, Ebru S Selen, Michael J Wolfgang, Steven A Farber, Peter J Espenshade.
      Oxygen regulates hypoxia-inducible factor (HIF) transcription factors to control cell metabolism, erythrogenesis, and angiogenesis. Whereas much has been elucidated about how oxygen regulates HIF, whether lipids affect HIF activity is un-known. Here, using cultured cells and two animal models, we demonstrate that lipoprotein-derived fatty acids are an independent regulator of HIF. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of the HIFα subunit of these heterodimeric transcription factors comparable with hypoxia with activation of downstream target genes. The addition of fatty acids to culture medium suppressed this signal, which required an intact mitochondrial respiratory chain. Mechanistically, fatty acids and oxygen are distinct signals integrated to control HIF activity. Finally, we observed lipid signaling to HIF and changes in target gene expression in developing zebrafish and adult mice, and this pathway operates in cancer cells from a range of tissues. This study identifies fatty acids as a physiological modulator of HIF, defining a mechanism for lipoprotein regulation that functions in parallel to oxygen.
    Keywords:  fatty acid; hypoxia-inducible factor (HIF); lipoprotein; low-density lipoprotein; low-density lipoprotein (LDL); lysosomal acid lipase; mitochondria
    DOI:  https://doi.org/10.1074/jbc.RA120.015238
  20. Nat Cancer. 2020 Oct;1(10): 998-1009
    Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase.
    Angela Maria Savino, Sara Isabel Fernandes, Orianne Olivares, Anna Zemlyansky, Antony Cousins, Elke K Markert, Shani Barel, Ifat Geron, Liron Frishman, Yehudit Birger, Cornelia Eckert, Sergey Tumanov, Gillian MacKay, Jurre J Kamphorst, Pawel Herzyk, Jonatan Fernández-García, Ifat Abramovich, Inbal Mor, Michela Bardini, Ersilia Barin, Sudha Janaki-Raman, Justin R Cross, Michael G Kharas, Eyal Gottlieb, Shai Izraeli, Christina Halsey.
      Metabolic reprogramming is a key hallmark of cancer, but less is known about metabolic plasticity of the same tumor at different sites. Here, we investigated the metabolic adaptation of leukemia in two different microenvironments, the bone marrow and the central nervous system (CNS). We identified a metabolic signature of fatty-acid synthesis in CNS leukemia, highlighting Stearoyl-CoA desaturase (SCD1) as a key player. In vivo SCD1 overexpression increases CNS disease, whilst genetic or pharmacological inhibition of SCD1 decreases CNS load. Overall, we demonstrated that leukemic cells dynamically rewire metabolic pathways to suit local conditions and that targeting these adaptations can be exploited therapeutically.
    Keywords:  SCD1; acute lymphoblastic leukemia; central nervous system; fatty acid synthesis; metabolic reprogramming
    DOI:  https://doi.org/10.1038/s43018-020-00115-2
  21. Metabolites. 2021 Jan 14. pii: E51. [Epub ahead of print]11(1):
    L-Carnitine and Acylcarnitines: Mitochondrial Biomarkers for Precision Medicine.
    Marc R McCann, Mery Vet George De la Rosa, Gus R Rosania, Kathleen A Stringer.
      Biomarker discovery and implementation are at the forefront of the precision medicine movement. Modern advances in the field of metabolomics afford the opportunity to readily identify new metabolite biomarkers across a wide array of disciplines. Many of the metabolites are derived from or directly reflective of mitochondrial metabolism. L-carnitine and acylcarnitines are established mitochondrial biomarkers used to screen neonates for a series of genetic disorders affecting fatty acid oxidation, known as the inborn errors of metabolism. However, L-carnitine and acylcarnitines are not routinely measured beyond this screening, despite the growing evidence that shows their clinical utility outside of these disorders. Measurements of the carnitine pool have been used to identify the disease and prognosticate mortality among disorders such as diabetes, sepsis, cancer, and heart failure, as well as identify subjects experiencing adverse drug reactions from various medications like valproic acid, clofazimine, zidovudine, cisplatin, propofol, and cyclosporine. The aim of this review is to collect and interpret the literature evidence supporting the clinical biomarker application of L-carnitine and acylcarnitines. Further study of these metabolites could ultimately provide mechanistic insights that guide therapeutic decisions and elucidate new pharmacologic targets.
    Keywords:  acyl-carnitine; metabolic flexibility; metabolism; metabolomics; mitochondria; pharmacometabolomics
    DOI:  https://doi.org/10.3390/metabo11010051
  22. Anal Chem. 2021 Jan 19.
    DaDIA: Hybridizing Data-Dependent and Data-Independent Acquisition Modes for Generating High-Quality Metabolomic Data.
    Jian Guo, Sam Shen, Shipei Xing, Tao Huan.
      Existing data acquisition modes such as full-scan, data-dependent (DDA), and data-independent acquisition (DIA) often present limited capabilities in capturing metabolic information in liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. In this work, we proposed a novel metabolomic data acquisition workflow that combines DDA and DIA analyses to achieve better metabolomic data quality, including enhanced metabolome coverage, tandem mass spectrometry (MS2) coverage, and MS2 quality. This workflow, named data-dependent-assisted data-independent acquisition (DaDIA), performs untargeted metabolomic analysis of individual biological samples using DIA mode and the pooled quality control (QC) samples using DDA mode. This combination takes advantage of the high-feature number and MS2 spectral coverage of the DIA data and the high MS2 spectral quality of the DDA data. To analyze the heterogeneous DDA and DIA data, we further developed a computational program, DaDIA.R, to automatically extract metabolic features and perform streamlined metabolite annotation of DaDIA data set. Using human urine samples, we demonstrated that the DaDIA workflow delivers remarkably improved data quality when compared to conventional DDA or DIA metabolomics. In particular, both the number of detected features and annotated metabolites were greatly increased. Further biological demonstration using a leukemia metabolomics study also proved that the DaDIA workflow can efficiently detect and annotate around 4 times more significant metabolites than DDA workflow with broad MS2 coverage and high MS2 spectral quality for downstream statistical analysis and biological interpretation. Overall, this work represents a critical development of data acquisition mode in untargeted metabolomics, which can greatly benefit untargeted metabolomics for a wide range of biological applications.
    DOI:  https://doi.org/10.1021/acs.analchem.0c05022
  23. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Dec 24. pii: S1570-0232(20)31391-X. [Epub ahead of print]1164 122515
    A perspective on the standards describing mass spectrometry-based metabolic phenotyping (metabolomics/metabonomics) studies in publications.
    Ian D Wilson, Georgios Theodoridis, Christina Virgiliou.
      As metabolic phenotyping (metabolomics, metabonomics and also lipidomics) gains in popularity and new investigators enter the field, the need to maintain and improve standards in publication is ever more pressing. In this perspective the requirements for information that should be included in manuscripts published in the Journal of Chromatography B, to ensure that the work is both credible and repeatable, are discussed. These include aspects such as study design, ethics, quality assurance (QA), quality control (QC) and data processing. In addition, aspects such as the level of confidence required for reporting metabolite identification (to a level where they could be subsequently used to develop hypotheses) are discussed.
    Keywords:  Data analysis; GC–MS; LC–MS; Mass spectrometry; Metabolite identification; Quality assurance; Quality control; Reporting standards
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122515
  24. Cancers (Basel). 2021 Jan 05. pii: E147. [Epub ahead of print]13(1):
    Human Plasma Metabolomics for Biomarker Discovery: Targeting the Molecular Subtypes in Breast Cancer.
    Leticia Díaz-Beltrán, Carmen González-Olmedo, Natalia Luque-Caro, Caridad Díaz, Ariadna Martín-Blázquez, Mónica Fernández-Navarro, Ana Laura Ortega-Granados, Fernando Gálvez-Montosa, Francisca Vicente, José Pérez Del Palacio, Pedro Sánchez-Rovira.
       PURPOSE: The aim of this study is to identify differential metabolomic signatures in plasma samples of distinct subtypes of breast cancer patients that could be used in clinical practice as diagnostic biomarkers for these molecular phenotypes and to provide a more individualized and accurate therapeutic procedure.
    METHODS: Untargeted LC-HRMS metabolomics approach in positive and negative electrospray ionization mode was used to analyze plasma samples from LA, LB, HER2+ and TN breast cancer patients and healthy controls in order to determine specific metabolomic profiles through univariate and multivariate statistical data analysis.
    RESULTS: We tentatively identified altered metabolites displaying concentration variations among the four breast cancer molecular subtypes. We found a biomarker panel of 5 candidates in LA, 7 in LB, 5 in HER2 and 3 in TN that were able to discriminate each breast cancer subtype with a false discovery range corrected p-value < 0.05 and a fold-change cutoff value > 1.3. The model clinical value was evaluated with the AUROC, providing diagnostic capacities above 0.85.
    CONCLUSION: Our study identifies metabolic profiling differences in molecular phenotypes of breast cancer. This may represent a key step towards therapy improvement in personalized medicine and prioritization of tailored therapeutic intervention strategies.
    Keywords:  breast cancer; human plasma metabolomics; metabolic profiling; molecular subtypes; personalized medicine
    DOI:  https://doi.org/10.3390/cancers13010147
  25. Proteomics. 2021 Jan 17. e2000140
    Advances in quantitative high-throughput phosphoproteomics with sample multiplexing.
    Joao A Paulo, Devin K Schweppe.
      Protein phosphorylation modulates nearly every major biological process. Phosphorylation regulates protein activity, mediates cellular signal transduction, and manipulates cellular structure. Consequently, the dysregulation of kinase and phosphatase pathways has been linked to a multitude of diseases. Mass spectrometry-based proteomic techniques are increasingly used for the global interrogation of perturbations in phosphorylation-based cellular signaling. Strategies for studying phosphoproteomes require high-specificity enrichment, sensitive detection, and accurate localization of phosphorylation sites with advanced LC-MS/MS techniques and downstream informatics. Sample multiplexing with isobaric tags has also been integral to recent advancements in throughput and sensitivity for phosphoproteomic studies. Each of these facets of phosphoproteomics analysis present distinct challenges and thus opportunities for improvement and innovation. Here, we review current methodologies, explore persistent challenges, and discuss the outlook for isobaric tag-based quantitative phosphoproteomic analysis. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1002/pmic.202000140
  26. Angew Chem Int Ed Engl. 2021 Jan 18.
    Imaging and analysis of isomeric unsaturated lipids through online photochemical derivatization of C=C bonds.
    Julia Laskin, Daisy Unsihuay, Pei Su, Hang Hu, Jiamin Qiu, Shihuan Kuang, Yingju Li, Xiaofei Sun, Sudhansu K Dey.
      Unraveling the complexity of the lipidome requires the development of novel approaches for the structural characterization of lipid species with isomer-level discrimination. Herein, we introduce an online photochemical approach for lipid isomer identification through selective derivatization of double bonds by reaction with singlet oxygen. Lipid hydroperoxide products are generated promptly after laser irradiation. Fragmentation of these species in a mass spectrometer produces diagnostic fragments, which reveal the C=C locations in the unreacted lipids. This approach uses an inexpensive light source and photosensitizer making it easy to incorporate into any lipidomics workflow. We demonstrate the utility of this approach for the shotgun profiling of C=C locations in different lipid classes present in tissue extracts using electrospray ionization (ESI) and ambient imaging of lipid species differing only by the location of C=C bonds using nanospray desorption electrospray ionization (nano-DESI).
    Keywords:  double bond position; lipid oxidation; photochemistry; singlet oxygen; unsaturated lipids
    DOI:  https://doi.org/10.1002/anie.202016734
  27. Mol Cell Proteomics. 2021 Jan 13. pii: S1535-9476(21)00001-3. [Epub ahead of print] 100046
    Ethical Principles, Constraints and Opportunities in Clinical Proteomics.
    Sebastian Porsdam Mann, Peter V Treit, Philipp E Geyer, Gilbert S Omenn, Matthias Mann.
      Recent advances in mass spectrometry (MS)-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with impacts on the health, privacy, and wellbeing of individuals. We conducted and here report a systematic literature review of ethical issues in clinical proteomics. We add our perspectives from a background of bioethics, the results of our accompanying paper extracting individual-sensitive results from patient samples, and the literature addressing similar issues in genomics. The spectrum of potential issues ranges from patient re-identification to incidental findings of clinical significance. The latter can be divided into actionable and unactionable findings. Some of these have the potential to be employed in discriminatory or privacy-infringing ways. However, incidental findings may also have great positive potential. A plasma proteome profile, for instance, could inform on the general health or disease status of an individual regardless of the narrow diagnostic question that prompted it. We suggest that early discussion of ethical issues in clinical proteomics can ensure that eventual healthcare practices and regulations reflect the considered judgment of the community and anticipate opportunities and problems that may arise as the technology matures.
    DOI:  https://doi.org/10.1016/j.mcpro.2021.100046
  28. Anal Chem. 2021 Jan 22.
    An Ion Chromatography-Ultrahigh-Resolution-MS1/Data-Independent High-Resolution MS2 Method for Stable Isotope-Resolved Metabolomics Reconstruction of Central Metabolic Networks.
    Qiushi Sun, Teresa W-M Fan, Andrew N Lane, Richard M Higashi.
      The metabolome comprises a complex network of interconnecting enzyme-catalyzed reactions that involve transfers of numerous molecular subunits. Thus, the reconstruction of metabolic networks requires metabolite substructures to be tracked. Subunit tracking can be achieved by tracing stable isotopes through metabolic transformations using NMR and ultrahigh -resolution (UHR)-mass spectrometry (MS). UHR-MS1 readily resolves and counts isotopic labels in metabolites but requires tandem MS to help identify isotopic enrichment in substructures. However, it is challenging to perform chromatography-based UHR-MS1 with its long acquisition time, while acquiring MS2 data on many coeluting labeled isotopologues for each metabolite. We have developed an ion chromatography (IC)-UHR-MS1/data-independent(DI)-HR-MS2 method to trace the fate of 13C atoms from [13C6]-glucose ([13C6]-Glc) in 3D A549 spheroids in response to anticancer selenite and simultaneously 13C/15N atoms from [13C5,15N2]-glutamine ([13C5,15N2]-Gln) in 2D BEAS-2B cells in response to arsenite transformation. This method retains the complete isotopologue distributions of metabolites via UHR-MS1 while simultaneously acquiring substructure label information via DI-MS2. These details in metabolite labeling patterns greatly facilitate rigorous reconstruction of multiple, intersecting metabolic pathways of central metabolism, which are illustrated here for the purine/pyrimidine nucleotide biosynthesis. The pathways reconstructed based on subunit-level isotopologue analysis further reveal specific enzyme-catalyzed reactions that are impacted by selenite or arsenite treatments.
    DOI:  https://doi.org/10.1021/acs.analchem.0c03070
  29. J Biol Chem. 2020 Nov 27. pii: S0021-9258(20)00094-0. [Epub ahead of print]296 100104
    ABHD5 suppresses cancer cell anabolism through lipolysis-dependent activation of the AMPK/mTORC1 pathway.
    Guohua Chen, Guoli Zhou, Aaron Lotvola, James G Granneman, Jian Wang.
      ABHD5 is an essential coactivator of ATGL, the rate-limiting triglyceride (TG) lipase in many cell types. Importantly, ABHD5 also functions as a tumor suppressor, and ABHD5 mRNA expression levels correlate with patient survival for several cancers. Nevertheless, the mechanisms involved in ABHD5-dependent tumor suppression are not known. We found that overexpression of ABHD5 induces cell cycle arrest at the G1 phase and causes growth retardation in a panel of prostate cancer cells. Transcriptomic profiling and biochemical analysis revealed that genetic or pharmacological activation of lipolysis by ABHD5 potently inhibits mTORC1 signaling, leading to a significant downregulation of protein synthesis. Mechanistically, we found that ABHD5 elevates intracellular AMP content, which activates AMPK, leading to inhibition of mTORC1. Interestingly, ABHD5-dependent suppression of mTORC1 was abrogated by pharmacological inhibition of DGAT1 or DGAT2, isoenzymes that re-esterify fatty acids in a process that consumes ATP. Collectively, this study maps out a novel molecular pathway crucial for limiting cancer cell proliferation, in which ABHD5-mediated lipolysis creates an energy-consuming futile cycle between TG hydrolysis and resynthesis, leading to inhibition of mTORC1 and cancer cell growth arrest.
    Keywords:  AMP-activated protein kinase (AMPK); cancer metabolism; lipolysis; mTOR; αβ hydrolase domain containing 5 (ABHD5)
    DOI:  https://doi.org/10.1074/jbc.RA120.014682
  30. Biomolecules. 2021 Jan 15. pii: E113. [Epub ahead of print]11(1):
    Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.
    Laura Niederstaetter, Benjamin Neuditschko, Julia Brunmair, Lukas Janker, Andrea Bileck, Giorgia Del Favero, Christopher Gerner.
      Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.
    Keywords:  batch variations; eicosanoids; fetal calf serum; mass spectrometry; peroxisomes; proteomics
    DOI:  https://doi.org/10.3390/biom11010113
  31. Methods Mol Biol. 2021 ;2251 157-175
    Exploring Phosphoinositide Binding Using Native Mass Spectrometry.
    Julian Bender, Carla Schmidt.
      Phosphoinositides interact with proteins to fulfill various functions in the cell. In many cases, they specifically recruit peripheral membrane proteins to biological membranes. The analysis of their interactions with proteins is therefore essential for understanding the underlying processes. Native mass spectrometry (MS) preserves noncovalent interactions in the gas phase of a mass spectrometer and is therefore well-suited to study protein-phosphoinositide interactions. In this protocol, we describe the application of native MS to integral and peripheral membrane proteins and their interactions with lipids. We discuss sample and instrumental requirements, the realization of experiments, and the data analysis workflow. We further describe a biochemical assay to proof interactions of peripheral membrane proteins with lipids.
    Keywords:  Membrane proteins; Native mass spectrometry; Phosphoinositides; Protein–phosphoinositide interactions
    DOI:  https://doi.org/10.1007/978-1-0716-1142-5_11
  32. Acta Neuropathol Commun. 2021 Jan 19. 9(1): 16
    Glioma cells require one-carbon metabolism to survive glutamine starvation.
    Kazuhiro Tanaka, Takashi Sasayama, Hiroaki Nagashima, Yasuhiro Irino, Masatomo Takahashi, Yoshihiro Izumi, Takiko Uno, Naoko Satoh, Akane Kitta, Katsusuke Kyotani, Yuichi Fujita, Mitsuru Hashiguchi, Tomoaki Nakai, Masaaki Kohta, Yoichi Uozumi, Masakazu Shinohara, Kohkichi Hosoda, Takeshi Bamba, Eiji Kohmura.
      Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from nutrient starvation. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in GBM cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around "pseudopalisading necrosis." Genetic suppression of MTHFD2 and autophagy inhibition caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation. These results highlight a critical role for serine-dependent one-carbon metabolism in surviving glutamine starvation and suggest new therapeutic targets for glioma cells adapting to a low-nutrient microenvironment.
    Keywords:  Glioblastoma multiforme; Glutamine starvation; One-carbon metabolism; Serine synthesis
    DOI:  https://doi.org/10.1186/s40478-020-01114-1
  33. Metabolomics. 2021 Jan 18. 17(2): 14
    Metabolic consequences for mice lacking Endosialin: LC-MS/MS-based metabolic phenotyping of serum from C56Bl/6J Control and CD248 knock-out mice.
    Emily G Armitage, Alan Barnes, Kieran Patrick, Janak Bechar, Matthew J Harrison, Gareth G Lavery, G Ed Rainger, Christopher D Buckley, Neil J Loftus, Ian D Wilson, Amy J Naylor.
       INTRODUCTION: The Endosialin/CD248/TEM1 protein is expressed in adipose tissue and its expression increases with obesity. Recently, genetic deletion of CD248 has been shown to protect mice against atherosclerosis on a high fat diet.
    OBJECTIVES: We investigated the effect of high fat diet feeding on visceral fat pads and circulating lipid profiles in CD248 knockout mice compared to controls.
    METHODS: From 10 weeks old, CD248-/- and +/+ mice were fed either chow (normal) diet or a high fat diet for 13 weeks. After 13 weeks the metabolic profiles and relative quantities of circulating lipid species were assessed using ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry (UHPLC-MS) with high resolution accurate mass (HRAM) capability.
    RESULTS: We demonstrate a specific reduction in the size of the perirenal fat pad in CD248-/- mice compared to CD248+/+, despite similar food intake. More strikingly, we identify significant, diet-dependent differences in the serum metabolic phenotypes of CD248 null compared to age and sex-matched wildtype control mice. Generalised protection from HFD-induced lipid accumulation was observed in CD248 null mice compared to wildtype, with particular reduction noted in the lysophosphatidylcholines, phosphatidylcholines, cholesterol and carnitine.
    CONCLUSIONS: Overall these results show a clear and protective metabolic consequence of CD248 deletion in mice, implicating CD248 in lipid metabolism or trafficking and opening new avenues for further investigation using anti-CD248 targeting agents.
    Keywords:  CD248; Endosialin; HRAM UHPLC–MS/MS; High fat diet
    DOI:  https://doi.org/10.1007/s11306-020-01764-1
  34. J Proteomics. 2021 Jan 13. pii: S1874-3919(21)00016-6. [Epub ahead of print] 104117
    Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues.
    Katarina Davalieva, Sanja Kiprijanovska, Aleksandar Dimovski, Gorazd Rosoklija, Andrew J Dwork.
      The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64-69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SIGNIFICANCE: Here we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE analysis.
    Keywords:  FASP; FFPE; LC-MS/MS; Label-free data-independent acquisition; Protein extraction; RapiGest
    DOI:  https://doi.org/10.1016/j.jprot.2021.104117
  35. Mol Metab. 2021 Jan 13. pii: S2212-8778(21)00005-3. [Epub ahead of print] 101165
    Nicotinamide N-methyltransferase: at the crossroads between cellular metabolism and epigenetic regulation.
    Annalisa Roberti, Agustín F Fernández, Mario F Fraga.
       BACKGROUND: The abundance of energy metabolites is intimately interconnected with the activity of chromatin modifying enzymes in order to guarantee the finely tuned modulation of gene expression in response to cellular energetic status. Metabolism-induced epigenetic gene regulation is a key molecular axis for the maintenance of cellular homeostasis and its deregulation is associated with several pathological conditions. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) using the universal methyl donor S-adenosyl methionine (SAM), directly linking one-carbon metabolism with a cell's methylation balance and nicotinamide adenine dinucleotide (NAD+) levels. NNMT expression and activity are regulated in a tissue-specific-manner and the protein can act either physiologically or pathologically depending on its distribution. While in the liver NNMT exerts a beneficial effect by regulating lipid parameters, in adipose tissue its expression correlates with obesity and insulin resistance. NNMT upregulation has been observed in a variety of cancers, and increased NNMT expression has been associated with tumor progression, metastasis and worse clinical outcomes. Accordingly, NNMT represents an appealing druggable target for metabolic disorders as well as oncological and other disease where the protein is improperly activated.
    SCOPE OF REVIEW: This review examines emerging findings concerning the complex NNMT regulatory network and the role of NNMT in both NAD metabolism and cell methylation balance. We extensively describe recent findings concerning the physiological and pathological regulation of NNMT with a specific focus on the function of NNMT in obesity, insulin resistance and other associated metabolic disorders along with its well-accepted role as a cancer-associated metabolic enzyme. Advances in strategies targeting NNMT pathways are also reported, together with current limitations of NNMT inhibitor drugs in clinical use.
    MAJOR CONCLUSIONS: NNMT is emerging as a key point of intersection between cellular metabolism and epigenetic gene regulation and growing evidence supports its central role in several pathologies. The use of molecules that target NNMT represents a current pharmaceutical challenge for the treatment of several metabolic-related disease as well as in cancer.
    Keywords:  Nicotinamide N-methyltransferase; cancer; epigenetics; metabolism; obesity
    DOI:  https://doi.org/10.1016/j.molmet.2021.101165
  36. Cancer Res. 2021 Jan 22. pii: canres.3477.2020. [Epub ahead of print]
    Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer.
    Ali Ghoochani, En-Chi Hsu, Merve Aslan, Meghan A Rice, Holly M Nguyen, James D Brooks, Eva Corey, Ramasamy Paulmurugan, Tanya Stoyanova.
      Ferroptosis is a type of programmed cell death induced by the accumulation of lipid peroxidation and lipid reactive oxygen species (ROS) in cells. It has been recently demonstrated that cancer cells are vulnerable to ferroptosis inducers (FIN). However, the therapeutic potential of ferroptosis inducers in prostate cancer in pre-clinical settings has not been explored. In this study, we demonstrate that mediators of ferroptosis SLC7A11, SLC3A2 and GPX4 are expressed in treatment-resistant prostate cancer. We further demonstrate that treatment-resistant prostate cancer cells are sensitive to two ferroptosis inducers, erastin and RSL3. Treatment with erastin and RSL3 led to a significant decrease in prostate cancer cell growth and migration in vitro and significantly delayed the tumor growth of treatment-resistant prostate cancer in vivo, with no measurable side effects. Combination of erastin or RSL3 with standard-of-care second-generation anti-androgens for advanced prostate cancer halted prostate cancer cell growth and migration in vitro and tumor growth in vivo. These results demonstrate the potential of erastin or RSL3 independently and in combination with standard-of-care second-generation anti-androgens as novel therapeutic strategies for advanced prostate cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3477
  37. Methods Mol Biol. 2021 ;2251 19-37
    Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.
    Avishek Ghosh, Padinjat Raghu.
      Phosphoinositides (PIs), the seven phosphorylated derivatives of phosphatidylinositol, are recognized as key molecules in the control of multiple molecular events in eukaryotic cells. Within cells, PIs are low-abundance lipids making their detection and quantification challenging. While many methods that allow radiolabeling and quantification of PIs in the context of cultured cells are available, these are not useful in the context of in vivo animal models where cell and developmental processes are best studied. In this chapter, we describe radionuclide-free, mass spectrometry-based methods for the detection and quantification of PIs from Drosophila tissues in vivo. The use of these methods should facilitate the discovery of novel modes by which PIs regulate cellular and developmental processes in complex metazoans.
    Keywords:  Drosophila; In vivo biochemistry; Mass spectrometry; Phosphoinositides; label-free quantification
    DOI:  https://doi.org/10.1007/978-1-0716-1142-5_2
  38. Cancer Metab. 2021 Jan 22. 9(1): 5
    Pharmacological activation of pyruvate kinase M2 reprograms glycolysis leading to TXNIP depletion and AMPK activation in breast cancer cells.
    Fadi Almouhanna, Biljana Blagojevic, Suzan Can, Ali Ghanem, Stefan Wölfl.
       BACKGROUND: Aerobic glycolysis, discovered by Otto Warburg, is a hallmark of cancer metabolism even though not yet fully understood. The low activity of the cancerous pyruvate kinase isozyme (M2) is thought to play an important role by facilitating the conversion of glycolytic intermediates to other anabolic pathways to support tumors' high proliferation rate.
    METHODS: Five breast cancer cell lines representing different molecular subtypes were used in this study where real time measurements of cellular bioenergetics and immunoblotting analysis of energy- and nutrient-sensing pathways were employed to investigate the potential effects of PKM2 allosteric activator (DASA-58) in glucose rewiring.
    RESULTS: In this study, we show that DASA-58 can induce pyruvate kinase activity in breast cancer cells without affecting the overall cell survival. The drug is also able to reduce TXNIP levels (an intracellular glucose sensor) probably through depletion of upstream glycolytic metabolites and independent of AMPK and ER signaling. AMPK shows an induction in phosphorylation (T172) in response to treatment an effect that can be potentiated by combining DASA-58 with other metabolic inhibitors.
    CONCLUSIONS: Altogether, the multifaceted metabolic reprogramming induced by DASA-58 in breast cancer cells increases their susceptibility to other therapeutics suggesting the suitability of the intracellular glucose sensor TXNIP as a marker of PK activity.
    Keywords:  AMPK; Breast cancer; Cancer metabolism; Glycolysis; Pyruvate kinase M2; TXNIP
    DOI:  https://doi.org/10.1186/s40170-021-00239-8
  39. Drug Metab Pharmacokinet. 2021 Jan 14. pii: S1347-4367(20)30438-9. [Epub ahead of print]37 100377
    Application of comprehensive lipidomics to biomarker research on adverse drug reactions.
    Kosuke Saito.
      Lipidomics is a relatively new field of omics that focuses on lipids, one of the major categories of metabolites. Owing to their various functions, lipids are considered suitable targets for biomarker development; in addition, lipidomics analysis of adverse drug reactions (ADRs) has been conducted recently. In this review, I have summarized information on comprehensive lipidomics, which involves the analysis of global lipids in a non-targeted manner. Mass spectrometry-based platforms are currently the dominant lipidomics platform owing to their versatile features. I have also summarized the application of lipidomics in biomarker research on ADRs caused by therapeutic drugs in humans and rodents. Additionally, general concerns in and emerging approaches of lipidomics research on ADR have been highlighted. Although biomarkers identified using the lipidomics analysis of ADRs have not been qualified, reported candidates will be evaluated for clinical application. In addition, novel biomarker candidates will be developed via classical and new approaches exemplified in this review.
    Keywords:  Adverse drug reaction; Biomarker; Lipid; Lipidomics; Liver injury; Mass spectrometry
    DOI:  https://doi.org/10.1016/j.dmpk.2020.100377
  40. J Biol Chem. 2020 Aug 21. pii: S0021-9258(17)50076-9. [Epub ahead of print]295(34): 12167-12180
    A functional role for eicosanoid-lysophospholipids in activating monocyte signaling.
    Gao-Yuan Liu, Sung Ho Moon, Christopher M Jenkins, Harold F Sims, Shaoping Guan, Richard W Gross.
      Recently, eicosanoid-lysophospholipids were identified as novel metabolites generated from the direct cyclooxygenase- or lipoxygenase-catalyzed oxidation of 2-arachidonoyl-lysophospholipids produced from either phospholipase A1-mediated hydrolysis of diacyl arachidonoyl-phospholipids or through the cytochrome c-catalyzed oxidative hydrolysis of the vinyl ether linkage of arachidonoyl-plasmalogens. Although the metabolic pathways generating eicosanoid-lysophospholipids have been increasingly appreciated, the signaling functions of eicosanoid-lysophospholipids remain largely unknown. Herein, we demonstrate that 2-12(S)-HETE-lysophospholipids as well as nonesterified 12(S)-HETE are potent lipid mediators that activate THP-1 human monocytic cells to generate tumor necrosis factor α (TNFα) and interleukin 8 (IL8). Remarkably, low nanomolar concentrations of 12(S)-HETE-lysophospholipids, but not other oxidized signaling lipids examined activated THP-1 cells resulting in the production of large amounts of TNFα. Moreover, TNFα release induced by 12(S)-HETE-lysophospholipids was inhibited by the TNFα converting enzyme inhibitor TAPI-0 indicating normal processing of TNFα in THP-1 cells stimulated with these agonists. Western blotting analyses revealed that 12(S)-HETE-lysophospholipids activated the phosphorylation of NFκB p65, suggesting activation of the canonical NFκB signaling pathway. Importantly, activation of THP-1 cells to release TNFα was stereoselective with 12(S)-HETE favored over 12(R)-HETE. Furthermore, the EC50 of 2-12(S)-HETE-lysophosphatidylcholine in activating THP-1 cells was 2.1 nm, whereas the EC50 of free 12(S)-HETE was 23 nm. Additionally, lipid extracts of activated platelets were separated by RP-HPLC demonstrating the coelution of 12(S)-HETE with fractions initiating TNFα release. Collectively, these results demonstrate the potent signaling properties of 2-12(S)-HETE-lysophospholipids and 12(S)-HETE by their ability to release TNFα and activate NFκB signaling thereby revealing a previously unknown role of 2-12(S)-HETE-lysophospholipids in mediating inflammatory responses.
    Keywords:  12(S)-HETE; NFκB; cytokine; i-PLA2γ; iPLA2γ; inflammation; lysophospholipid; oxidized lysophospholipids; phosphorylation
    DOI:  https://doi.org/10.1074/jbc.RA120.013619
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