bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2020‒11‒22
thirty-five papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Front Oncol. 2020 ;10 589508
      Metabolism rewiring is an important hallmark of cancers. Being one of the most abundant free amino acids in the human blood, glutamine supports bioenergetics and biosynthesis, tumor growth, and the production of antioxidants through glutaminolysis in cancers. In glutamine dependent cancer cells, more than half of the tricarboxylic/critic acid (TCA) metabolites are derived from glutamine. Glutaminolysis controls the process of converting glutamine into TCA cycle metabolites through the regulation of multiple enzymes, among which the glutaminase shows the importance as the very first step in this process. Targeting glutaminolysis via glutaminase inhibition emerges as a promising strategy to disrupt cancer metabolism and tumor progression. Here, we review the regulation of glutaminase and the role of glutaminase in cancer metabolism and metastasis. Furthermore, we highlight the glutaminase inhibitor based metabolic therapy strategy and their potential applications in clinical scenarios.
    Keywords:  cancer metabolism; combination therapy; glutaminase inhibitor; glutaminolysis; metastasis
  2. J Biol Chem. 2020 Nov 18. pii: jbc.RA120.014888. [Epub ahead of print]
      Normal human cells can either synthesize cholesterol or take it up from lipoproteins to meet their metabolic requirements. In some malignant cells, de novo cholesterol synthesis genes are transcriptionally silent or mutated, meaning that cholesterol uptake from lipoproteins is required for survival. Recent data suggest that lymphoma cells dependent upon lipoprotein-mediated cholesterol uptake are also subject to ferroptosis, an oxygen- and iron-dependent cell death mechanism triggered by accumulation of oxidized lipids in cell membranes unless the lipid hydroperoxidase, glutathione peroxidase 4 (GPX4), reduces these toxic lipid species. To study mechanisms linking cholesterol uptake with ferroptosis and determine the potential role of the high-density lipoprotein (HDL) receptor as a target for cholesterol depleting therapy, we treated lymphoma cell lines known to be sensitive to reduction of cholesterol uptake with HDL-like nanoparticles (HDL NPs). HDL NPs are a cholesterol-poor ligand that binds to the receptor for cholesterol-rich HDL, scavenger receptor type B-1 (SCARB1). Our data reveal that HDL NP treatment activates a compensatory metabolic response in treated cells towards increased de novo cholesterol synthesis, which is accompanied by nearly complete reduction in expression of GPX4. As a result, oxidized membrane lipids accumulate leading to cell death through a mechanism consistent with ferroptosis. We obtained similar results in vivo after systemic administration of HDL NPs in mouse lymphoma xenografts and in primary samples obtained from patients with lymphoma. In summary, targeting SCARB1 with HDL NPs in cholesterol uptake-addicted lymphoma cells abolishes GPX4 resulting in cancer cell death by a mechanism consistent with ferroptosis.
    Keywords:  Ferroptosis; cholesterol; cholesterol metabolism; glutathione peroxidase; high-density lipoprotein (HDL); lipid peroxidation; lymphoma; nanotechnology
  3. J Biol Chem. 2020 Nov 20. pii: jbc.RA120.014682. [Epub ahead of print]
      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:  lipid signaling; lipolysis; metabolic regulation; tumor cell biology; tumor metabolism
  4. Mol Cell Proteomics. 2020 Nov 17. pii: mcp.R120.002257. [Epub ahead of print]
      Histone post-translational modifications (PTMs) are one of the main mechanisms of epigenetic regulation. Dysregulation of histone PTMs leads to many human diseases, such as cancer. Due to its high-throughput, accuracy, and flexibility, mass spectrometry (MS) has emerged as a powerful tool in the epigenetic histone modification field, allowing the comprehensive and unbiased analysis of histone PTMs and chromatin-associated factors. Coupled with various techniques from molecular biology, biochemistry, chemical biology and biophysics, MS has been employed to characterize distinct aspects of histone PTMs in the epigenetic regulation of chromatin functions. In this review we will describe advancements in the field of MS that have facilitated the analysis of histone PTMs and chromatin biology.
    Keywords:  Affinity proteomics; Chemoproteomics; Epigenetics; Histones*; Mass Spectrometry; Post-translational modifications*
  5. Rapid Commun Mass Spectrom. 2020 Nov 17. e9007
      RATIONALE: Compound-specific isotope analysis (CSIA) of organic sulfur molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural-abundance sulfur isotopic compositions of the sulfur-containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields.METHODS: Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reverse-phase high performance liquid chromatography (HPLC) and verified via offline liquid chromatography mass spectrometry (LC/MS). The sulfur isotope ratios (δ34 S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature-ramped chromatographic column and programmable He carrier flow rates.
    RESULTS: On-column focusing of SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity allowing precise (0.1-0.3‰ 1s.d.) δ34 S measurements of 1 to 10 μg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof-of-concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ34 S values of cysteine and methionine that can be connected to biosynthetic pathways.
    CONCLUSIONS: We have developed a sensitive, precise method for measuring the natural-abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment to fundamental aspects of metabolism and biosynthesis.
  6. Expert Rev Proteomics. 2020 Nov 16.
      Introduction: We present our views on the current application of mass spectrometry (MS) based lipidomics and how lipidomics can develop in the next decade to be most practical use to society. That is not to say that lipidomics has not already been of value. In-fact, in its earlier guise as metabolite profiling most of the pathways of steroid biosynthesis were uncovered and via focused lipidomics many inborn errors of metabolism are routinely clinically identified. However, can lipidomics be extended to improve biochemical understanding of, and to diagnose, the most prevalent diseases of the 21st century? Areas covered: We will highlight the concept of "level of identification" and the equally crucial topic of "quantification". Only by using a standardised language for these terms can lipidomics be translated to fields beyond academia. We will remind the lipid scientist of the value of chemical derivatisation, a concept exploited since the dawn of lipid biochemistry. Expert opinion: Only by agreement of the concepts of identification and quantification and their incorporation in lipidomics reporting can lipidomics maximise its value.
    Keywords:  Clinical chemistry; identification; imaging; in-born errors of metabolism; lipids; mass spectrometry; medicine; quantification
  7. iScience. 2020 Nov 20. 23(11): 101703
      Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.
    Keywords:  Cellular Neuroscience; Lipidomics; Metabolomics; Molecular Neuroscience
  8. Cells. 2020 Nov 16. pii: E2491. [Epub ahead of print]9(11):
      Liver cancer is one of the most common cancer worldwide with a high mortality. Methionine is an essential amino acid required for normal development and cell growth, is mainly metabolized in the liver, and its role as an anti-cancer supplement is still controversial. Here, we evaluate the effects of methionine supplementation in liver cancer cells. An integrative proteomic and metabolomic analysis indicates a rewiring of the central carbon metabolism, with an upregulation of the tricarboxylic acid (TCA) cycle and mitochondrial adenosine triphosphate (ATP) production in the presence of high methionine and AMP-activated protein kinase (AMPK) inhibition. Methionine supplementation also reduces growth rate in liver cancer cells and induces the activation of both the AMPK and mTOR pathways. Interestingly, in high methionine concentration, inhibition of AMPK strongly impairs cell growth, cell migration, and colony formation, indicating the main role of AMPK in the control of liver cancer phenotypes. Therefore, regulation of methionine in the diet combined with AMPK inhibition could reduce liver cancer progression.
    Keywords:  AMPK; HCC; TCA cycle; growth; metabolomics; migration; proteomics
  9. J Biomol Tech. 2020 Aug;31(Suppl): S20-S21
      There is increasing need for throughput as the metabolomics studies are getting larger. Throughput can be achieved on the analytical side by using rapid methods or speeding up the data analysis and metabolite identification steps. Series of rapid UPLC-MS/MS methods have been developed on a single platform with identical analysis workflow for high throughput measurement of derivatized amino acids, acylcarnitines, bile acids, free fatty acids, tryptophan metabolites in human serum to support metabolomics research. The separation of isomers (amino acids and bile acids) are achieved in analytical runtimes of <4mins making these methods powerful and are well suited for a Core laboratory. Here we discuss these methods and demonstrate their usability for the analysis of metabolites in patient derived serum samples during targeted multi-omics analysis. Sample preparation involved protein precipitation with methanol (1:4 serum:methanol) for the extraction of acylcarnitines, bile acids, and free fatty acids. For amino acid analysis, serum samples were prepared using the Waters™ AccQTag Kit following the Kit protocol. Tryptophan metabolites sample preparation was achieved using Oasis HLB PRiME µElution Plate. UPLC separation was performed on an ACQUITY UPLC I-Class System (fixed loop), equipped with a CORTECS T3 2.7 µm (2.1 x 30 mm) analytical column. A 2 µ Lextract was injected at a flow rate of 1.3 mL/min. Mobile phase A was 0.01% formic acid (aq) and Mobile phase B was 50% isopropanol in acetonitrile containing 0.01% formic acid. The LC gradient and column equilibration times were optimized for each class of metabolites. The analytical column temperature was maintained at 60°C. Multiple Reaction Monitoring (MRM) analyses were performed using a Xevo TQ-S micro mass spectrometer. All experiments were performed in electrospray ionization mode. Data processing was done using in TargetLynx and Skyline.
  10. Metabolites. 2020 Nov 15. pii: E464. [Epub ahead of print]10(11):
      Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.
    Keywords:  high-resolution mass spectrometry; human plasma; ion mobility mass spectrometry; metabolite annotation; metabolomics; tandem mass spectrometry; ultra-high performance liquid chromatography
  11. Molecules. 2020 Nov 13. pii: E5307. [Epub ahead of print]25(22):
      Obesity is associated with alterations in the composition and amounts of lipids. Lipids have over 1.7 million representatives. Most lipid groups differ in composition, properties and chemical structure. These small molecules control various metabolic pathways, determine the metabolism of other compounds and are substrates for the syntheses of different derivatives. Recently, lipidomics has become an important branch of medical/clinical sciences similar to proteomics and genomics. Due to the much higher lipid accumulation in obese patients and many alterations in the compositions of various groups of lipids, the methods used for sample preparations for lipidomic studies of samples from obese subjects sometimes have to be modified. Appropriate sample preparation methods allow for the identification of a wide range of analytes by advanced analytical methods, including mass spectrometry. This is especially the case in studies with obese subjects, as the amounts of some lipids are much higher, others are present in trace amounts, and obese subjects have some specific alterations of the lipid profile. As a result, it is best to use a method previously tested on samples from obese subjects. However, most of these methods can be also used in healthy, nonobese subjects or patients with other dyslipidemias. This review is an overview of sample preparation methods for analysis as one of the major critical steps in the overall analytical procedure.
    Keywords:  biological samples; lipids; liquid–liquid extraction; obesity; protein precipitation; sample preparation; solid-phase extraction
  12. Metabolites. 2020 Nov 12. pii: E457. [Epub ahead of print]10(11):
      Reliable analyte identification is critical in metabolomics experiments to ensure proper interpretation of data. Due to chemical similarity of metabolites (as isobars and isomers) identification by mass spectrometry or chromatography alone can be difficult. Here we show that isomeric compounds are quite common in the metabolic space as given in common metabolite databases. Further, we show that retention information can shift dramatically between different experiments decreasing the value of external or even in-house compound databases. As a consequence the retention information in compound databases should be updated regularly, to allow a reliable identification. To do so we present a feasible and budget conscious method to guarantee updates of retention information on a regular basis using well designed compound mixtures. For this we combine compounds in "Ident-Mixes", showing a way to distinctly identify chemically similar compounds through combinatorics and principle of exclusion. We illustrate the feasibility of this approach by comparing Gas chromatography (GC)-columns with identical properties from three different vendors and by creating a compound database from measuring these mixtures by Liquid chromatography-mass spectrometry (LC-MS). The results show the high influence of used materials on retention behavior and the ability of our approach to generate high quality identifications in a short time.
    Keywords:  GC–MS; LC–MS; chromatography; identification; metabolomics; retention index; standardization
  13. Biochim Biophys Acta Mol Basis Dis. 2020 Nov 16. pii: S0925-4439(20)30359-8. [Epub ahead of print] 166011
      Abnormal glucose metabolism in cancer cells causes generation and secretion of excess lactate, which results in acidification of the extracellular microenvironment. This altered metabolism aids not only in survival and proliferation but also in suppressing immune-mediated destruction of cancer cells. However, how it influences the response of cancer cells to chemotherapeutic drugs is not clearly understood. We employed appropriate in vitro approaches to explore the role of mono-carboxylate transporter 4 (MCT4) mediated altered intra and extracellular pH on the outcome of the therapeutic efficacy of chemotherapeutic drugs in breast and lung cancer models. We demonstrate by in vitro experiments that inhibition of complex I enhance glycolysis and increases expression as well as membrane translocation of MCT4. It causes a decrease in extracellular pH (pHe) and impairs doxorubicin and paclitaxel's therapeutic efficacy. Acidic pHe inhibits doxorubicin's uptake, while acidic intracellular pH (pHi) impairs the efficacy of paclitaxel. Under in vivo experimental settings, the modulation of pHe with phloretin or alkalizer (NaHCO3) enhances cytotoxicity of drugs and inhibits the growth of MCF-7 xenografts in mice. In a nutshell, this study indicates that MCT4 mediated extracellular acidosis is involved in impairing chemotherapeutic drugs' efficacy on cancer cells. Therefore, the use of pH neutralizing agents or MCT inhibitors may be beneficial towards circumventing impairment in the efficacy of certain drugs that are sensitive to pH changes.
    Keywords:  Chemotherapeutic outcome; Lactic acidosis; MCTs; Metabolic reprogramming; Metformin; Tumor-microenvironment
  14. Oncogene. 2020 Nov 20.
      Prostate cancer is responsible for over 30,000 US deaths annually, attributed largely to incurable metastatic disease. Here, we demonstrate that high levels of plectin are associated with localized and metastatic human prostate cancer when compared to benign prostate tissues. Knock-down of plectin inhibits prostate cancer cell growth and colony formation in vitro, and growth of prostate cancer xenografts in vivo. Plectin knock-down further impairs aggressive and invasive cellular behavior assessed by migration, invasion, and wound healing in vitro. Consistently, plectin knock-down cells have impaired metastatic colonization to distant sites including liver, lung, kidney, bone, and genitourinary system. Plectin knock-down inhibited number of metastases per organ, as well as decreased overall metastatic burden. To gain insights into the role of plectin in prostate cancer growth and metastasis, we performed proteomic analysis of prostate cancer plectin knock-down xenograft tissues. Gene set enrichment analysis shows an increase in levels of proteins involved with extracellular matrix and laminin interactions, and a decrease in levels of proteins regulating amino acid metabolism, cytoskeletal proteins, and cellular response to stress. Collectively these findings demonstrate that plectin is an important regulator of prostate cancer cell growth and metastasis.
  15. J Mass Spectrom. 2020 Nov 18. e4681
      Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 μl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 μg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.
    Keywords:  MRM profiling; direct injection; exploratory lipidomics; extracellular vesicles; lipid profiling
  16. Mol Aspects Med. 2020 Nov 17. pii: S0098-2997(20)30135-7. [Epub ahead of print] 100933
      Colorectal cancer (CRC) is one of the most commonly occurring cancers worldwide. Although several genetic alterations have been associated with CRC onset and progression, nowadays the reprogramming of cellular metabolism has been recognized as a fundamental step of the carcinogenic process. Intestinal tumor cells frequently display an aberrant activation of lipid metabolism. Indeed, to satisfy the growing needs of a continuous proliferation, cancer cells can either increase the uptake of exogenous lipids or upregulate the endogenous lipogenesis and cholesterol synthesis. Therefore, strategies aimed at limiting lipid accumulation are now under development in order to counteract malignancies. Two major players of lipids metabolism have been so far identified for their contribution to CRC development: the nuclear receptor Liver X Receptor (LXRs) and the enzyme Stearoyl-CoA Desaturase 1 (SCD1). Whereas LXR is mainly recognized for its role as a cholesterol sensor, finally promoting the loss of cellular cholesterol and whole-body homeostasis, SCD1 acts as the major regulator of new fatty acids, finely tuning the monounsaturated fatty acids (MUFA) to saturated fatty acids (SFA) ratio. Intriguingly, SCD1 is directly regulated by LXRs. Despite LXRs agonists have elicited great interest as a promising therapeutic target for cancer, LXR's ability to induce SCD1 and new fatty acids synthesis represent a major obstacle in the development of new effective treatments. Thus, further investigations are required to fully dissect the concomitant modulation of both players, to develop specific therapies aimed at blocking intestinal cancer cells proliferation, eventually counteracting CRC progression.
    Keywords:  Colorectal cancer; Lipid metabolism; Liver X receptor; Nuclear receptor; Stearoyl-CoA Desaturase
  17. Cell Metab. 2020 Oct 24. pii: S1550-4131(20)30544-1. [Epub ahead of print]
      Acute or chronic cellular stress resulting from aberrant metabolic and biochemical processes may trigger a pervasive non-apoptotic form of cell death, generally known as ferroptosis. Ferroptosis is unique among the different cell death modalities, as it has been mostly linked to pathophysiological conditions and because several metabolic pathways, such as (seleno)thiol metabolism, fatty acid metabolism, iron handling, mevalonate pathway, and mitochondrial respiration, directly impinge on the cells' sensitivity toward lipid peroxidation and ferroptosis. Additionally, key cellular redox systems, such as selenium-dependent glutathione peroxidase 4 and the NAD(P)H/ferroptosis suppressor protein-1/ubiquinone axis, are at play that constantly surveil and neutralize oxidative damage to cellular membranes. Since this form of cell death emerges to be the root cause of a number of diseases and since it offers various pharmacologically tractable nodes for therapeutic intervention, there has been overwhelming interest in the last few years aiming for a better molecular understanding of the ferroptotic death process.
    Keywords:  FSP1; GPX4; ferroptosis; lipid peroxidation; redox metabolism
  18. Metabolomics. 2020 Nov 20. 16(12): 121
      BACKGROUND: Dietary patterns low in glycemic load are associated with reduced risk of cardiometabolic diseases. Improvements in serum lipid concentrations may play a role in these observed associations.OBJECTIVE: We investigated how dietary patterns differing in glycemic load affect clinical lipid panel measures and plasma lipidomics profiles.
    METHODS: In a crossover, controlled feeding study, 80 healthy participants (n = 40 men, n = 40 women), 18-45 y were randomized to receive low-glycemic load (LGL) or high glycemic load (HGL) diets for 28 days each with at least a 28-day washout period between controlled diets. Fasting plasma samples were collected at baseline and end of each diet period. Lipids on a clinical panel including total-, VLDL-, LDL-, and HDL-cholesterol and triglycerides were measured using an auto-analyzer. Lipidomics analysis using mass-spectrometry provided the concentrations of 863 species. Linear mixed models and lipid ontology enrichment analysis were implemented.
    RESULTS: Lipids from the clinical panel were not significantly different between diets. Univariate analysis showed that 67 species on the lipidomics panel, predominantly in the triacylglycerol class, were higher after the LGL diet compared to the HGL (FDR < 0.05). Three species with FA 17:0 were lower after LGL diet with enrichment analysis (FDR < 0.05).
    CONCLUSION: In the context of controlled eucaloric diets with similar macronutrient distribution, these results suggest that there are relative shifts in lipid species, but the overall pool does not change. Further studies are needed to better understand in which compartment the different lipid species are transported in blood, and how these shifts are related to health outcomes. This trial was registered at as NCT00622661.
    Keywords:  Dietary pattern; High glycemic load; Low glycemic load; Randomized crossover feeding study; Targeted lipidomics
  19. Endocrine. 2020 Nov 21.
      PURPOSE: To review the literature assessing the diagnostic performance of urinary steroid profiling (USP) by high-performance liquid chromatography (LC-MS) or gas chromatography (GC) coupled to mass spectrometry (MS) in the evaluation of adrenal lesions, both in terms of functionality and malignancy.RESULTS: The evaluation of adrenal incidentalomas (AI) aims to rule out malignancy and hormone excess. Current diagnostic protocols have several limitations and include time consuming and relatively complicated multi-step processes in most cases. On the contrary, USP by LC-MS/MS or LC-GC/MS offer an easy, comprehensive and non-invasive assessment of adrenal steroid secretion. USP complements current workups used in the evaluation of AIs by improving our ability to identify malignancy and/or autonomous hormone secretion.
    CONCLUSIONS: Urine steroid profiling by LC-MS/MS and GC-MS allows a thorough, non-invasive, assessment of adrenal steroidogenesis as a whole which complement the current evaluation of AIs, and holds a promising role in the diagnosis of autonomous cortisol secretion, primary aldosteronism, and adrenal malignancy.
    Keywords:  Adrenal incidentalomas; Autonomous cortisol secretion; Mass spectrometry; Tetra-hydrocortisol; Tetrahydro-11-deoxycortisol; Urine steroid profile
  20. Int J Mol Sci. 2020 Nov 16. pii: E8623. [Epub ahead of print]21(22):
      The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every continent, registering over 1,250,000 deaths worldwide. The effects of SARS-CoV-2 on host targets remains largely limited, hampering our understanding of Coronavirus Disease 2019 (COVID-19) pathogenesis and the development of therapeutic strategies. The present study used a comprehensive untargeted metabolomic and lipidomic approach to capture the host response to SARS-CoV-2 infection. We found that several circulating lipids acted as potential biomarkers, such as phosphatidylcholine 14:0_22:6 (area under the curve (AUC) = 0.96), phosphatidylcholine 16:1_22:6 (AUC = 0.97), and phosphatidylethanolamine 18:1_20:4 (AUC = 0.94). Furthermore, triglycerides and free fatty acids, especially arachidonic acid (AUC = 0.99) and oleic acid (AUC = 0.98), were well correlated to the severity of the disease. An untargeted analysis of non-critical COVID-19 patients identified a strong alteration of lipids and a perturbation of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, aminoacyl-tRNA degradation, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. The severity of the disease was characterized by the activation of gluconeogenesis and the metabolism of porphyrins, which play a crucial role in the progress of the infection. In addition, our study provided further evidence for considering phospholipase A2 (PLA2) activity as a potential key factor in the pathogenesis of COVID-19 and a possible therapeutic target. To date, the present study provides the largest untargeted metabolomics and lipidomics analysis of plasma from COVID-19 patients and control groups, identifying new mechanisms associated with the host response to COVID-19, potential plasma biomarkers, and therapeutic targets.
    Keywords:  SARS-CoV-2; biomarkers; fatty acids; metabolism
  21. Anal Chim Acta. 2020 Dec 01. pii: S0003-2670(20)30913-2. [Epub ahead of print]1139 88-99
      Lipidomics focuses on the comprehensive analysis of lipids and their interactions with other molecules. Many biological samples are available in small volumes or limited amounts and display very complex lipid compositions; thus, highly sensitive lipidomic analysis methods are often needed. NanoLC-MS offers extremely high sensitivity, although it is known to be technically more challenging than the conventional LC-MS approach, requiring greater care and maintenance. This work describes the development and optimization of a nanoLC-MS method for routine analysis of the lipidome of small volumes of biological samples. We focused on achieving robust conditions for high sensitivity analysis of complex samples. The nanoLC method, mass spectrometry conditions and sample preparation by liquid-liquid extraction of lipids were fully optimized using serum samples and deuterated lipid standards, including an evaluation of contamination sources. The performance of the method was assessed through the analysis of human and pig sera, as well as cerebrospinal fluid samples from pigs. This method allowed the detection of 9900 to 12,200 features by employing only 1.0-2.5 μL of serum samples and identification of 5842 lipids from 36 subclasses within a 50-min gradient. The method can be easily adapted to other types of biological samples where only limited volumes are available.
    Keywords:  Biological samples; Lipidomics; Mass spectrometry; Sensitivity; nanoLC
  22. Front Cell Dev Biol. 2020 ;8 597608
      Tumor progression is a complex process consisting of several steps characterized by alterations in cellular behavior and morphology. These steps include uncontrolled cell division and proliferation, invasiveness and metastatic ability. Throughout these phases, cancer cells encounter a changing environment and a variety of metabolic stress. To meet their needs for energy while they proliferate and survive in their new environment, tumor cells need to continuously fine-tune their metabolism. The connection between intracellular transport and metabolic reprogramming during cancer progression is emerging as a central process of cellular adaptation to these changes. The trafficking of proteolytic enzymes, surface receptors, but also the regulation of downstream pathways, are all central to cancer progression. In this review, we summarize different hallmarks of cancer with a special focus on the role of intracellular trafficking in cell proliferation, epithelial to mesenchymal transition as well as invasion. We will further emphasize how intracellular trafficking contributes to the regulation of energy consumption and metabolism during these steps of cancer progression.
    Keywords:  cancer cell metabolism; cell proliferation; epithelial to mesenchymal transition; invasion; membrane trafficking
  23. Methods Mol Biol. 2021 ;2230 449-456
      Radiolabeled amino acid uptake assays are a highly sensitive method used to characterize the uptake of amino acids by cells or tissues in culture. This method is an excellent tool to quantify changes in amino acid consumption that are associated with states of cellular differentiation and/or disease. The methods presented here can be adapted to measure the transport of all amino acids and can be applied to cultured cells and bone explants.
    Keywords:  3H; Amino acid uptake; Bone explants; Primary cells; Radiolabeled isotopes; Tritium
  24. Methods Mol Biol. 2021 ;2230 437-447
      Measuring cellular metabolism accurately is necessary to understand bioenergetic pathways in cells. The major ATP generating pathways in cells are oxidative phosphorylation and glycolysis. We have recently analyzed and published bioenergetic pathways active in osteoblasts undergoing differentiation in response to various substrates. Based on those studies, here we provide step-by-step procedures to isolate, culture, plate and run a seahorse assay for measuring cellular metabolism. Furthermore, we provide an example of oxygen consumption and extracellular acidification rate traces obtained from MC3T3E1-C4 cells using the XFe96 seahorse analyzer. One of the limitations of studying bioenergetics in bone cells is the current lack of techniques to analyze bioenergetics in vivo in live animals. There are currently techniques that have been developed using third harmonic generation to study osteocytes using three-photon microscopy along with metabolic changes using endogenous two-photon excited fluorescence. However, these sophisticated techniques are not widely available. The relative ease with which one can obtain data pertaining to metabolic parameters using the XF technology makes it a very attractive technique to utilize on a monolayer of adherent cells.
    Keywords:  Bone marrow stromal cells; Calvarial osteoblasts; Extracellular acidification rate; Glycolysis; MC3T3E1C4 preosteoblast oxidative phosphorylation; Oxygen consumption rate
  25. Nat Chem Biol. 2020 Nov 16.
      Untargeted mass spectrometry is employed to detect small molecules in complex biospecimens, generating data that are difficult to interpret. We developed Qemistree, a data exploration strategy based on the hierarchical organization of molecular fingerprints predicted from fragmentation spectra. Qemistree allows mass spectrometry data to be represented in the context of sample metadata and chemical ontologies. By expressing molecular relationships as a tree, we can apply ecological tools that are designed to analyze and visualize the relatedness of DNA sequences to metabolomics data. Here we demonstrate the use of tree-guided data exploration tools to compare metabolomics samples across different experimental conditions such as chromatographic shifts. Additionally, we leverage a tree representation to visualize chemical diversity in a heterogeneous collection of samples. The Qemistree software pipeline is freely available to the microbiome and metabolomics communities in the form of a QIIME2 plugin, and a global natural products social molecular networking workflow.
  26. Metabolomics. 2020 Nov 18. 16(12): 120
      INTRODUCTION: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. An increasing number of studies have demonstrated that tumor proliferation and metastasis are closely related to complex metabolic reprogramming. However, there are limited data to provide a comprehensive metabolic picture of osteosarcoma.OBJECTIVES: Our study aims to identify aberrant metabolic pathways and seek potential adjuvant biomarkers for osteosarcoma.
    METHODS: Serum samples were collected from 65 osteosarcoma patients and 30 healthy controls. Nontargeted metabolomic profiling was performed by liquid chromatography-mass spectrometry (LC-MS) based on univariate and multivariate statistical analyses.
    RESULTS: The OPLS-DA model analysis identified clear separations among groups. We identified a set of differential metabolites such as higher serum levels of adenosine-5-monophosphate, inosine-5-monophosphate and guanosine monophosphate in primary OS patients compared to healthy controls, and higher serum levels of 5-aminopentanamide, 13(S)-HpOTrE (FA 18:3 + 2O) and methionine sulfoxide in lung metastatic OS patients compared to primary OS patients, revealing aberrant metabolic features during the proliferation and metastasis of osteosarcoma. We found a group of metabolites especially lactic acid and glutamic acid, with AUC values of 0.97 and 0.98, which could serve as potential adjuvant diagnostic biomarkers for primary osteosarcoma, and a panel of 2 metabolites, 5-aminopentanamide and 13(S)-HpOTrE (FA 18:3 + 2O), with an AUC value of 0.92, that had good monitoring ability for lung metastases.
    CONCLUSIONS: Our study provides new insight into the aberrant metabolic features of osteosarcoma. The potential biomarkers identified here may have translational significance.
    Keywords:  Biomarker; LC-MS; Metabolomics; Osteosarcoma
  27. Cancer Res. 2020 Nov 17. pii: canres.1517.2020. [Epub ahead of print]
      Alteration in lipid composition is an important metabolic adaptation by cancer cells to support tumorigenesis and metastasis. Fatty acid 2-hydroxylase (FA2H) introduces a chiral hydroxyl group at the second carbon of FA backbones and influences lipid structures and metabolic signaling. However, the underlying mechanisms through which FA 2-hydroxylation is coupled to metabolic adaptation and tumor growth remain elusive. Here we show that FA2H regulates specific metabolic reprogramming and oncogenic signaling in the development of colorectal cancer (CRC). FA2H was highly expressed in normal colorectal tissues. Assessments through deciphering both published high-throughput data and curated human CRC samples revealed significant suppression of FA2H in tumors, which is correlated with unfavorable prognosis. Experiments with multiple models of genetic manipulation or treatment with an enzymatic product of FA2H, (R)-2-hydroxy palmitic acid ((R)-2-OHPA), demonstrated that FA 2-hydroxylation inhibits CRC cell proliferation, migration, epithelial-mesenchymal transition (EMT) progression and tumor growth. Bioinformatics analysis suggested that FA2H functions through AMP-activated protein kinase/Yes-associated protein (AMPK/YAP) pathway, which was confirmed in CRC cells as well as in tumors. Lipidomics analysis revealed an accumulation of polyunsaturated fatty acids (PUFA) in cells with FA2H overexpression, which may contribute to the observed nutrient deficiency and AMPK activation. Collectively, these data demonstrate that FA 2-hydroxylation initiates a metabolic signaling cascade to suppress colorectal tumor growth and metastasis via the YAP transcriptional axis and provides a strategy to improve CRC treatment.
  28. Clin Rheumatol. 2020 Nov 20.
      Osteoarthritis (OA) represents the most prevalent and disabling arthritis worldwide due to its heterogeneous and progressive articular degradation. However, effective and timely diagnosis and fundamental treatment for this disorder are lacking. Metabolomics, a growing field in life science research in recent years, has the potential to detect many metabolites and thus explains the underlying pathophysiological processes. Hence, new specific metabolic markers and related metabolic pathways can be identified for OA. In this review, we aimed to provide an overview of studies related to the metabolomics of OA in animal models and humans to describe the metabolic changes and related pathways for OA. The present metabolomics studies reveal that the pathogenesis of OA may be significantly related to perturbations of amino acid metabolism. These altered amino acids (e.g., branched-chain amino acids, arginine, and alanine), as well as phospholipids, were identified as potential biomarkers to distinguish patients with OA from healthy individuals.
    Keywords:  Biomarkers; Metabolic pathways; Metabolomics; Osteoarthritis
  29. Anal Chim Acta. 2020 Dec 15. pii: S0003-2670(20)31040-0. [Epub ahead of print]1140 210-218
      The identification and quantification of biomarkers is essential for the diagnosis, treatment, and long-term monitoring of many human diseases. In the present work, macromolecular synthetic receptors with pre-determined affinity and selectivity for the signature peptide of a prognostically significant small cell lung cancer (SCLC) biomarker - neuron-specific enolase (NSE) - were prepared in a porous polymer microsphere format using a template-directed synthesis strategy performed under precipitation polymerization conditions. The polymer microspheres were packed into short trap columns and then exploited as molecularly selective sorbents in a fully automated, on-line molecularly imprinted solid-phase extraction (MISPE) protocol. The on-line MISPE protocol was optimised with respect to the composition of the loading mobile phase, the flow rate, and the extraction time. The molecularly imprinted polymers (MIPs) showed high affinity and useful selectivity for the peptide target - the hexapeptide ELPLYR - compared to non-imprinted control polymers. The MIPs were able to retain the biomarker on-column for extraction times of up to 20 min, and the on-line MISPE method enabled complete recovery of the biomarker over the linear range 10-100 ng mL-1 when the biomarker was present in spiked ammonium bicarbonate solution (R2 = 0.994). For extractions of ELPLYR from very complex biological matrices, the recoveries of ELPLYR from reversed-phase SPE (RP-SPE)-treated and untreated digested human serum were 100.8 ± 6.2% and 61.6 ± 1.9%, respectively. Extractions of ELPLYR from spiked untreated digested serum were linear in the range of 7.5-375 ng mL-1 (R2 = 0.99). The limit of detection (LOD) and limit of quantification (LOQ) for the biomarker in digested serum were estimated to be 1.8 ng mL-1 and 6.0 ng mL-1, respectively, which is below the median reference level of NSE in humans (8.6 ng mL-1). This work sets in place the basis for a new diagnostic tool for SCLC that is sensitive, robust, automated, and antibody-free, and which works very well with complex human plasma samples.
    Keywords:  Bottom-up proteomics; Liquid chromatography tandem mass spectrometry; Low-abundant biomarkers; Molecularly imprinted polymers; On-line solid-phase extraction
  30. ACS Med Chem Lett. 2020 Nov 12. 11(11): 2108-2113
      Mass spectrometry (MS) detection can offer unmatched selectivity and sensitivity. The use of MS without chromatography greatly increases the throughput, making it suitable for high throughput screening. However, the trade-offs of direct MS detection need to be carefully evaluated along with the development of novel strategies to ensure successful implementation. In this review, we will discuss the pros and cons of chromatography-free MS and discuss some of the currently used and future technologies being investigated to enable high-throughput MS.
  31. J Chromatogr A. 2020 Nov 05. pii: S0021-9673(20)30953-5. [Epub ahead of print]1634 461679
      The quantification of metabolites in various samples, including body fluids, tissues, cells, and foodstuffs, contributes to our understanding of their biological activities and roles in the body, diagnosis for many diseases, drug and biomarker discovery, and many aspects of human health. Liquid chromatography (LC)/tandem mass spectrometry (MS/MS) is the most powerful and reliable methodology for the quantification of metabolites due to its high specificity and sensitivity, and broad coverage of various compounds. Derivatization often makes the quantification power of LC/MS/MS stronger due to the desirable LC behavior and enhanced MS/MS detectability of the derivatized metabolites. On the other hand, LC/MS/MS-based quantification has room for improvement regarding its analysis throughput. Derivatization is also a promising approach to overcome this drawback; the multiplexing of samples in the same LC/MS/MS injection, which is achieved by derivatization of multiple samples with multiple well-designed reagents, can enhance the throughput. Based on this background, this article reviews the derivatization-based sample-multiplexing strategy, especially the characteristics and applications of the derivatization reagents, for the LC/MS/MS quantification of metabolites. This strategy has been used for the relative and absolute quantification of a variety of metabolites, and expansion of the coverage of metabolites.
    Keywords:  Derivatization; LC/MS/MS; Metabolite; Quantification; Sample-multiplexing; Throughput
  32. Nat Metab. 2020 Nov;2(11): 1284-1304
      Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.
  33. Anal Biochem. 2020 Nov 11. pii: S0003-2697(20)30548-0. [Epub ahead of print] 114016
      A one-vial extraction method for the quantitation of short-chain fatty acids (SCFAs) in human stool was developed. Samples were extracted with an acidified aqueous internal standard solution, sodium sulfate, and diethyl ether, followed by analysis with GC-FID. Accuracy, in terms of relative recovery, was typically between 90 and 110% for most analytes; without internal standard, the accuracy was about 5-34%; the linear dynamic range (LDR) was 0.05 to 50 micromoles per gram; the limit of detection (LOD) was less than or equal to 0.05 micromoles per gram; and the (lower) limit of quantitation (LOQ) was 1 micromole per gram. The method is suitable for quantitating acetic acid, propanoic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isohexanoic acid, hexanoic acid, and heptanoic acid. It is not suitable for the quantitation of formic acid. Application to human biological research was tested by the measurement of SCFA in heathy humans. This confirmed that the method performed adequately, and even better than expected, with values up to 150 micromoles per gram.
    Keywords:  Gas Chromatography; Gut Health; Human Stool; Short-Chain Fatty Acids
  34. ACS Omega. 2020 Nov 10. 5(44): 28877-28888
      RATIONALE: The low molecular weight (LMW) proteins present in circulating body fluids, such as serum and plasma, hold biological significance as possible biomarkers. A major obstacle in mass spectrometry-based proteomics of serum is the presence of abundant high molecular weight proteins which mask the identification and quantitation of lower molecular weight proteins. Traditional methods involve the use of affinity resins to remove high molecular weight proteins, such as albumin and immunoglobulin G, with concomitant loss of lower molecular weight proteins. Considering the importance of depleting high molecular proteins, this paper compares an affinity resin, a gel-filter, and an acetonitrile (ACN) precipitation method to achieve successful removal of high molecular weight proteins and recovery of lower molecular weight proteins.METHODS: Serum enrichment was carried out by multiple methods such as with the commercially available serum protein mini kit, ACN precipitation, and a gel filter method. Mass spectrometric runs were carried out on an AB SCIEX ESI QTOF 5600 mass spectrometer. Mass spectrometry analysis of the enriched serum obtained by ACN precipitation and gel filter method was performed for global proteome profiling. Quantitative mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ) for ACN-precipitated enriched serum was also carried out.
    RESULTS: The gel filter method, though allowing for the resolution and identification of LMW proteins, was better suited for global proteome analysis and not preferred for quantitative proteomic experiments. In contrast, enrichment by the ACN precipitation method allowed for the reproducible identification and quantitation of LMW proteins having molecular weight ≥4 kDa.
    CONCLUSIONS: Using only chilled ACN and centrifugation, most of the highly abundant proteins were successfully removed from the serum, while recovering a significant portion of the LMW proteome. A more rapid protocol, which is compatible with iTRAQ labeling, to achieve improved results has been elucidated, thus allowing for better screening and identification of potential biomarkers.
  35. J Proteome Res. 2020 Nov 14.
      The isobaric carrier approach, which combines small isobarically labeled samples with a larger isobarically labeled carrier sample, finds diverse applications in ultrasensitive mass spectrometry analysis of very small samples, such as single cells. To enhance the growing use of isobaric carriers, we characterized the trade-offs of using isobaric carriers in controlled experiments with complex human proteomes. The data indicate that isobaric carriers directly enhance peptide sequence identification without simultaneously increasing the number of protein copies sampled from small samples. The results also indicate strategies for optimizing the amount of isobaric carrier and analytical parameters, such as ion accumulation time, for different priorities such as improved quantification or an increased number of identified proteins. Balancing these trade-offs enables adapting isobaric carrier experiments to different applications, such as quantifying proteins from limited biopsies or organoids, building single-cell atlases, or modeling protein networks in single cells. In all cases, the reliability of protein quantification should be estimated and incorporated in all subsequent analyses. We expect that these guidelines will aid in explicit incorporation of the characterized trade-offs in experimental designs and transparent error propagation in data analysis.
    Keywords:  benchmarking; data reliability; isobaric carrier; optimizing mass spectrometry analysis; quantification accuracy; single-cell proteomics