bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019‒11‒17
twenty-four papers selected by
Giovanny Rodriguez Blanco
The Beatson Institute for Cancer Research

  1. Methods Mol Biol. 2020 ;2084 269-282
      Untargeted lipidomics aims to comprehensively measure and characterize all lipid species in biological systems. Ion mobility-mass spectrometry (IM-MS) has showed a great potential for untargeted lipidomic analysis. Coupling with liquid chromatography and data-independent tandem MS techniques, acquired IM-MS data set contains four-dimensional information for lipid identification, including m/z of MS1 ion, retention time (RT), collision cross section (CCS), and MS/MS spectra. In this protocol, we introduced a data processing workflow using an integrative web server, namely, LipidIMMS Analyzer, to support accurate lipid identification. The protocol demonstrated the integration of all four dimensional information to achieve unambiguous identifications of lipids in complex biological samples.
    Keywords:  Collision cross section; Ion mobility-mass spectrometry; Lipid identification; LipidIMMS Analyzer; Untargeted lipidomics
  2. Methods Mol Biol. 2020 ;2084 235-244
      Ambient ionization-based mass spectrometry (MS) methods coupled with ion mobility separation (IMS) have emerged as promising approaches for high-throughput in situ analysis for biomedical to environmental applications. These methods are capable of direct profiling and molecular imaging of metabolites, lipids, peptides, and xenobiotics from biological tissues with minimal sample preparation. Furthermore, employing IMS within the workflow improves the molecular coverage, resolves isobaric species, and improves biomolecule identifications through accurate collision cross section measurements. Laser ablation electrospray ionization (LAESI)-MS coupled with IMS has been successful in profiling and molecular imaging of small biomolecules directly from biological tissues and single cells. Herein, we describe a protocol for the direct analysis of adherent mammalian cells with limited perturbations by LAESI-IMS-MS. A benefit of IMS is that within the same LAESI acquisition, the spectral features related to the ESI background, washing buffer, and cell signal can be extracted and isolated separately.
    Keywords:  Cell culture; In situ analysis; Ion mobility separation; LAESI-IMS-MS; Laser ablation electrospray ionization; Mass spectrometry; Metabolites
  3. Methods Mol Biol. 2020 ;2077 225-235
      Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) is key for the characterization of phosphorylation sites in a high-throughput manner, and its application has proven essential to elucidate the phosphoproteome of many biological systems. Following proteolytic digestion of proteins extracted from tissues or cells, phosphopeptides are typically enriched by affinity chromatography using TiO2 or metal-ions (e.g., Fe3+) coupled to solid-phase materials, prior to LC-MS/MS analysis. Separation of relatively low abundance phosphopeptides from nonphosphorylated peptides in these types of extremely complex mixtures is essential to maximize coverage of the phosphoproteome. Maintaining acidic conditions during these IMAC or TiO2-based enrichment minimizes the concurrent unwanted binding of highly acidic peptides. However, while peptides containing phosphomonoesters, namely, phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr), are stable under these acidic binding conditions, phosphopeptides containing acid-labile phosphate group such as phosphohistidine (pHis), are not. Consequently, hydrolysis of these types of phosphopeptides occurs during standard phosphopeptide enrichment, and subsequent phosphosite identification by LC-MS/MS is severely compromised. Here we describe UPAX, unbiased phosphopeptide enrichment using strong anion exchange, for the separation of both acid-stable (pSer, pThr, pTyr) and acid-labile phosphopeptides (including those containing pHis) from nonphosphorylated peptides. We outline how implementation of UPAX prior to a minimally modified standard proteomics workflow can be used to identify sites of pHis as well as other acid-labile, as well as acid-stable phosphosites.
    Keywords:  Enrichment; Mass spectrometry; Phosphohistidine; Phosphoproteomics; Strong anion exchange; pHis
  4. Methods Mol Biol. 2020 ;2084 55-78
      Mass spectrometry-based metabolomics is being increasingly applied to a number of applications, including the fields of clinical, industrial, plant, and nutritional science. Several improvements have advanced the field considerably over the past decade, including ultra-high performance liquid chromatography (uHPLC), column chemistries, instruments, software, and molecular databases. However, challenges remain, including how to separate small molecules that are part of highly complex samples; this can be accomplished using chromatographic techniques or through improved resolution in the gas phase. Ion mobility-mass spectrometry (IM-MS) provides an extra dimension of gas phase separation that can result in improvements to both quantitation and compound identification. Here we describe a typical drift tube IM-MS metabolomics workflow, which includes the following steps: (1) Data acquisition, (2) Data preprocessing, (3) Molecular feature finding, and (4) Differential analysis and Molecular annotation. Overall, these methods can help investigators from a variety of scientific fields use IM-MS metabolomics as part of their own workflow.
    Keywords:  Acquisition parameters; Differential analysis; Drift tube ion mobility; Four-dimensional feature finding; Mass spectrometry; Metabolomics
  5. Methods Mol Biol. 2020 ;2084 79-94
      This chapter describes the developments in drift-tube ion mobility-mass spectrometry (DTIM-MS) that have driven application development in 'omics analyses. Harnessing the additional, orthogonal separation that DTIM provides increased confidence in compound identifications as the mass spectral complexity can be reduced and mobility-derived parameters (most prominently the collision cross section, CCS) used to support identity confirmation goals for a variety of 'omics application areas. Presented within this contribution is a methodology for improving the transmission and maintaining accurate determination of drift time-derived CCS (DTCCS) for low molecular weight compounds for a typical nontargeted 'omics (metabolomics) workflow using liquid chromatography in combination with DTIM-MS.
    Keywords:  CCS databases; Collision cross section; Drift-tube ion mobility-mass spectrometry; Ion mobility; Mass spectrometry; Metabolomics; Non-targeted analysis; Small molecules
  6. Methods Mol Biol. 2020 ;2084 35-54
      Metabolites and xenobiotics are small molecules with a molecular weight that often falls below 600 Da. Over the last few decades, multiple small molecule databases have been curated listing structures, masses, and fragmentation spectra possible in metabolomic and exposomic measurements. To date only a small portion of the spectra in these databases are experimentally derived due to the high expense of obtaining, synthesizing, and analyzing standards. A vast majority of spectra have thus been created using theoretical programs to fit the available experimental data. The errors associated with theoretical data have however caused problems with current small molecule identifications, and accurate quantitation as searching the databases using just one or two analysis dimensions (i.e., chromatography retention times and mass spectrometry (MS) m/z values) results in numerous annotations for each experimental feature. Additional analysis dimensions are therefore needed to better annotate and identify small molecules. Drift tube ion mobility spectrometry coupled with MS (DTIMS-MS) is a promising technique to address this challenge as it is able to perform rapid structural evaluations of small molecules in complex matrices by assessing the collision cross section values for each in addition to their m/z values. The use of IMS in conjunction with other separation techniques such as gas or liquid chromatography and MS has therefore enabled more accurate identifications for the small molecules present in complex biological and environmental samples. Here, we present a review of relevant parameter considerations for DTIMS application with emphasis on xenobiotics and metabolomics isomer separations.
    Keywords:  Collision cross section (CCS); Drift tube ion mobility spectrometry (DTIMS); Exposomics; Ion mobility spectrometry (IMS); Metabolomics
  7. J Lipid Res. 2019 Nov 15. pii: jlr.D119000393. [Epub ahead of print]
      Quantitative MS of human plasma lipids is a promising technology for translation into clinical applications. Current MS-based lipidomic methods rely on either direct infusion or chromatographic lipid separation methods (including reversed-phase and hydrophilic interaction liquid chromatography). However, the use of lipid markers in laboratory medicine is limited by the lack of reference values, largely because of considerable differences in the concentrations measured by different laboratories worldwide. These inconsistencies can be explained by the use of different sample preparation protocols, method-specific calibration procedures, and other experimental and data-reporting parameters, even when using identical starting materials. Here, we systematically investigated the roles of some of these variables in multiple approaches to lipid analysis of plasma samples from healthy adults, by  considering (1) different sample introduction methods (separation vs. direct infusion methods), (2) different MS instruments and (3) between-laboratory differences in comparable analytical platforms. Each of these experimental variables resulted in different quantitative results, even with the inclusion of isotope-labelled internal standards for individual lipid classes. We demonstrated that appropriate normalization to commonly available reference samples (i.e., "shared references") can largely correct for these systematic, method-specific quantitative biases. Thus, to harmonize data in the field of lipidomics, in-house, long-term references should be complemented by a commonly available shared reference sample, such as NIST SRM 1950, in the case of human plasma.
    Keywords:  Harmonization; Lipidomics; Lipids; Liquid chromatography; Mass spectrometry; NIST SRM 1950; Phospholipids; Plasma; Quantitation; Sphingolipids
  8. Nephrology (Carlton). 2019 Nov 09.
      AIM: Laser microdissection (LMD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) enable clinicians to analyse proteins from tissue sections. In nephrology, these methods are used to diagnose diseases of abnormal protein deposition, such as amyloidosis, but they are seldom applied to the diagnosis and pathophysiological understanding of human glomerular diseases.METHODS: Renal biopsy specimens were obtained from five patients with IgA nephropathy (IgAN), five patients with membranous nephropathy (MN) and five kidney transplant donors (as controls). From 10-μm-thick sections of formalin-fixed, paraffin-embedded specimens, 0.3-mm2 samples of glomerular tissue were subjected to LMD. The samples were analysed by LC-MS/MS and investigated clinically and histologically.
    RESULTS: From the control glomeruli, we identified more than 300 types of proteins. In patients with IgAN, we detected significant increases not only in IgA1 and in C3 but also in the factors related to oxidative stress and cell proliferation in comparison to the controls. In patients with MN, levels of IgG1, IgG4, C3, C4a, and phospholipase-A2-receptor were significantly elevated in comparison to the controls, as were the aforementioned factors related to oxidative stress and cell proliferations detected in IgAN.
    CONCLUSION: Application of LMD and LC-MS/MS to renal biopsy specimens enabled us to identify not only pathognomonic proteins for the diagnosis but also several factors possibly involved in the pathogenesis of human glomerular diseases. This article is protected by copyright. All rights reserved.
    Keywords:  IgA nephropathy; glomerulonephritis; laser microdissection; mass spectrometry; membranous nephropathy
  9. Methods Mol Biol. 2020 ;2084 119-132
      Lipidomics is a rapidly growing field that enables the characterization of the entire lipidome in cells, tissues, or an organism. Changes in lipid metabolism and homeostasis caused by different disease states or drug treatments can be probed by lipidomics experiments, which can aid our understanding of normal physiology and disease pathology at the molecular level. While current technologies using liquid chromatography coupled with high-resolution mass spectrometry have greatly increased coverage of the lipidome, there are still limitations in resolving the large number of lipid species with similar masses in a narrow mass window. We recently reported that two orthogonal separation techniques, hydrophilic interaction liquid chromatography (HILIC) and ion mobility (IM), enhance the resolution of lipid species based on headgroup polarity and gas-phase size and shape, respectively, of various classes of glycerolipids, glycolipids, phospholipids, and sphingolipids. Here we describe the application of our HILIC-IM-MS lipidomics protocol to the analysis of lipid extracts derived from either tissues or cells, to identify significant changes in the lipidome in response to an internal or external stimulus, such as exposure to environmental chemicals.
    Keywords:  Collision cross section; Hydrophilic interaction liquid chromatography; Ion mobility; Lipidomics; Mass spectrometry
  10. J Lipid Res. 2019 Nov 11. pii: jlr.D119000318. [Epub ahead of print]
      Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, since it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are used often as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of 13C6-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with 13C6-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homologue of the calcium-independent phospholipase A2β, on lipid metabolism. Although 13C6-glucose gave rise to a complex pattern of 13C incorporation, we were able to identify discrete isotopomers in which 13C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.
    Keywords:  Genetics; Glycerolipids; Lipidomics; Mass spectrometry; Phospholipids/Metabolism
  11. Cells. 2019 Nov 12. pii: E1423. [Epub ahead of print]8(11):
      Hepatic fibrosis is a major cause of morbidity and mortality worldwide, as it ultimately leads to cirrhosis, which is estimated to affect up to 2% of the global population. Hepatic fibrosis is confirmed by liver biopsy, and the erroneous nature of this technique necessitates the search for noninvasive alternatives. However, current biomarker algorithms for hepatic fibrosis have many limitations. Given that the liver is the largest organ and a major metabolic hub in the body, probing the metabolic signature of hepatic fibrosis holds promise for the discovery of new markers and therapeutic targets. Regarding individual metabolic pathways, accumulating evidence shows that hepatic fibrosis leads to alterations in carbohydrate metabolism, as aerobic glycolysis is aggravated in activated hepatic stellate cells (HSCs) and the whole fibrotic liver; in amino acid metabolism, as Fischer's ratio (branched-chain amino acids/aromatic amino acids) decreases in patients with hepatic fibrosis; and in lipid metabolism, as HSCs lose vitamin A-containing lipid droplets during transdifferentiation, and cirrhotic patients have decreased serum lipids. The current review also summarizes recent findings of metabolic alterations relevant to hepatic fibrosis based on systems biology approaches, including transcriptomics, proteomics, and metabolomics in vitro, in animal models and in humans.
    Keywords:  Fischer’s ratio; HSC; TCA cycle; aerobic glycolysis; hepatic fibrosis; metabolomics; proteomics; transcriptomics
  12. Methods Mol Biol. 2020 ;2084 161-178
      Ion mobility combined with mass spectrometry (IM-MS) is a powerful technique for the analysis of biomolecules and complex mixtures. This chapter reviews the current state-of-the-art in ion mobility technology and its application to biology, protein analysis, and quantitative discovery proteomics in particular, from an analytical perspective. IM-MS can be used as a technique to separate mixtures, to determine structural information (rotationally averaged cross-sectional area) and to enhance MS duty cycle and sensitivity. Moreover, IM-MS is ideally suited for hyphenating with liquid chromatography, or other front-end separation techniques such as, GC, microcolumn LC, capillary electrophoresis, and direct analysis, including MALDI and DESI, providing an semiorthogonal layer of separation, which affords the more unambiguous and confident detection of a wide range of analytes. To illustrate these enhancements, as well as recent developments, the principle of in-line IM separation and hyphenation to orthogonal acceleration time-of-flight mass spectrometers are discussed, in addition to the enhancement of biophysical MS-based analysis using typical proteomics and related application examples.
    Keywords:  Cyclic TWIMS; Ion mobility-mass spectrometry; Protein analysis; Proteomics
  13. Annu Rev Physiol. 2019 Nov 15.
      Acidic metabolic waste products accumulate in the tumor microenvironment because of high metabolic activity and insufficient perfusion. In tumors, the acidity of the interstitial space and the relatively well-maintained intracellular pH influence cancer and stromal cell function, their mutual interplay, and their interactions with the extracellular matrix. Tumor pH is spatially and temporally heterogeneous, and the fitness advantage of cancer cells adapted to extracellular acidity is likely particularly evident when they encounter less acidic tumor regions, for instance, during invasion. Through complex effects on genetic stability, epigenetics, cellular metabolism, proliferation, and survival, the compartmentalized pH microenvironment favors cancer development. Cellular selection exacerbates the malignant phenotype, which is further enhanced by acid-induced cell motility, extracellular matrix degradation, attenuated immune responses, and modified cellular and intercellular signaling. In this review, we discuss how the acidity of the tumor microenvironment influences each stage in cancer development, from dysplasia to full-blown metastatic disease. Expected final online publication date for the Annual Review of Physiology, Volume 82 is February 10, 2020. Please see for revised estimates.
  14. Anal Chem. 2019 Nov 15.
      Sialylated N-glycans play pivotal role in several important biological and pathological processes. Their sialyl-linkage isomers, mostly α-2,3- and α-2,6-linked, act differently during the cellular events and several diseases. While mass spectrometry (MS) technology is a powerful tool in N-glycome analysis, it still suffers from inability to distinguish linkage isomers of native N-glycans. Herein, we described a sequential selective derivatization method, by which α-2,6- and α-2,3-linked sialic acids are sequentially labeled with methylamide incorporated with different sta-ble-isotope. Isobaric labeling avoids inducing bias in ionization efficiency and chromatographic behavior. In optimized reaction condition, high derivatization selectivity (~99%) was achieved for both α-2,3- and α-2,6-linked sialic acid. High accuracy of quantitation within dynamic range of two orders of magnitude and high reproducibility (CV<20%, n=3) were demonstrated using standard glycans and multi-sialylated N-glycans. Finally, this method was applied in profiling the N-glycome of serum from CRC patients, where level of six sialyl-linkage isomers were found to be altered significantly compared with that from healthy individuals.
  15. J Biol Chem. 2019 Nov 11. pii: jbc.RA119.011519. [Epub ahead of print]
      In cancer, aberrant growth factor receptor signaling reprograms cellular metabolism and global gene transcription to drive aggressive growth, but the underlying mechanisms are not well understood. Here we show that in the highly lethal brain tumor glioblastoma (GBM), mechanistic target of rapamycin complex 2 (mTORC2), a critical core component of the growth factor signaling system, couples acetyl-CoA production with nuclear translocation of histone-modifying enzymes including pyruvate dehydrogenase (PDH) and class IIa histone deacetylases (HDACs) to globally alter histone acetylation. Integrated analyses in orthotopic mouse models and in clinical GBM samples reveal that mTORC2 controls iron metabolisms via histone H3 acetylation of the iron-related gene promoter, promoting tumor cell survival. These results nominate mTORC2 as a critical epigenetic regulator of iron metabolism in cancer.
    Keywords:  acetyl coenzyme A (acetyl-CoA); glioblastoma; glucose metabolism; histone acetylation; iron metabolism; mammalian target of rapamycin (mTOR)
  16. Metabolites. 2019 Nov 12. pii: E276. [Epub ahead of print]9(11):
      Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities-particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies.
    Keywords:  biomarkers; diagnostics; exosomes; extracellular vesicles; metabolic pathways; microvesicles
  17. Metabolism. 2019 Nov 08. pii: S0026-0495(19)30220-3. [Epub ahead of print] 154005
      BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects 25-30% of the general population and is characterized by the presence of non-alcoholic fatty liver (NAFL) that can progress to non-alcoholic steatohepatitis (NASH), liver fibrosis and cirrhosis leading to hepatocellular carcinoma. To date, liver biopsy is the gold standard for the diagnosis of NASH and for staging liver fibrosis. This study aimed to train models for the non-invasive diagnosis of NASH and liver fibrosis based on measurements of lipids, glycans and biochemical parameters in peripheral blood and with the use of different machine learning methods.METHODS: We performed a lipidomic, glycomic and free fatty acid analysis in serum samples of 49 healthy subjects and 31 patients with biopsy-proven NAFLD (15 with NAFL and 16 with NASH). The data from the above measurements combined with measurements of 4 hormonal parameters were analyzed with two different platforms and five different machine learning tools.
    RESULTS: 365 lipids, 61 glycans and 23 fatty acids were identified with mass-spectrometry and liquid chromatography. Robust differences in the concentrations of specific lipid species were observed between healthy, NAFL and NASH subjects. One-vs-Rest (OvR) support vector machine (SVM) models with recursive feature elimination (RFE) including 29 lipids or combining lipids with glycans and/or hormones (20 or 10 variables total) could differentiate with very high accuracy (up to 90%) between the three conditions. In an exploratory analysis, a model consisting of 10 lipid species could robustly discriminate between the presence of liver fibrosis or not (98% accuracy).
    CONCLUSION: We propose novel models utilizing lipids, hormones and glycans that can diagnose with high accuracy the presence of NASH, NAFL or healthy status. Additionally, we report a combination of lipids that can diagnose the presence of liver fibrosis. Both models should be further trained prospectively and validated in large independent cohorts.
    Keywords:  Glycomics; Lipidomics; Liver fibrosis; Machine learning; Metabolomics; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Non-invasive
  18. Nat Protoc. 2019 Nov 13.
      Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology, MSI of tissue samples at single-cell resolution remains challenging. Herein, we describe a protocol for robust imaging of tissue sections with a high (better than 10-μm) spatial resolution using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry, an ambient ionization technique that does not require sample pretreatment before analysis. In this protocol, mouse uterine tissue is used as a model system to illustrate both the workflow and data obtained in these experiments. We provide a detailed description of the nano-DESI MSI platform, fabrication of the nano-DESI and shear force probes, shear force microscopy experiments, spectral acquisition, and data processing. A properly trained researcher (e.g., technician, graduate student, or postdoc) can complete all the steps from probe fabrication to data acquisition and processing within a single day. We also describe a new strategy for acquiring both positive- and negative-mode imaging data in the same experiment. This is achieved by alternating between positive and negative data acquisition modes during consecutive line scans. Using our imaging approach, hundreds of high-quality ion images were obtained from a single uterine section. This protocol enables sensitive and quantitative imaging of lipids and metabolites in heterogeneous tissue sections with high spatial resolution, which is critical to understanding biochemical processes occurring in biological tissues.
  19. Proteomics. 2019 Nov 15. e1900029
      An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for new and better biomarkers. Here, we review what leads to successful biomarker development and consider how these features may be applied in the context of proteomic biomarker research. The "fit-for-purpose" approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using mass spectrometry-based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. We discuss (i) the reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how mass spectrometry can improve, complement, or replace existing clinically-important assays in the future. This article is protected by copyright. All rights reserved.
    Keywords:  biomarker discovery; biomarker validation; clinical proteomics; mass spectrometry
  20. J Lipid Res. 2019 Nov 13. pii: jlr.RA119000256. [Epub ahead of print]
      Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine TNF-α are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5 - but not ACSL4 - in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.
    Keywords:  Acyl-CoA synthetase; Cytokines; Endothelial cells; Fatty acid/Metabolism; Fatty acid/Oxidation; Lipid droplets
  21. Methods Mol Biol. 2020 ;2084 1-31
      Ion mobility-mass spectrometry (IM-MS) combines complementary size- and mass-selective separations into a single analytical platform. This chapter provides context for both the instrumental arrangements and key application areas that are commonly encountered in bioanalytical settings. New advances in these high-throughput strategies are described with description of complementary informatics tools to effectively utilize these data-intensive measurements. Rapid separations such as these are especially important in systems, synthetic, and chemical biology in which many small molecules are transient and correspond to various biological classes for integrated omics measurements. This chapter highlights the fundamentals of IM-MS and its applications toward biomolecular separations and discusses methods currently being used in the fields of proteomics, lipidomics, and metabolomics.
    Keywords:  Biomolecules; Ion mobility; Ion mobility-mass spectrometry; Omics
  22. J Proteome Res. 2019 Nov 12.
      Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the brain as well as degeneration of motor and non-motor circuitry. The cause of neuronal death is currently unknown, although chronic neuroinflammation, aggregated α-synuclein, mitochondrial dysfunction and oxidative stress have all been implicated. Gliosis has been shown to exacerbate neuroinflammation via secretion of pro-inflammatory cytokines, and there is a subse-quent infiltration of T lymphocytes (T-cells), into the brain of PD patients. Using liquid chromatography-high resolution mass spectrometry (LC-HRMS), we have observed metabolomic changes in stool samples, thought to be associated with the potential disease-modifying effect of an immunotherapy administered to transgenic Parkinsonian (A53T) mice.
  23. Proc Natl Acad Sci U S A. 2019 Nov 15. pii: 201909243. [Epub ahead of print]
      Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression.
    Keywords:  NAD(P)H; extracellular vesicles; human breast cancer; in situ imaging; nonlinear microscopy
  24. Biochim Biophys Acta Mol Cell Res. 2019 Nov 07. pii: S0167-4889(19)30179-X. [Epub ahead of print] 118571
      The heterotrimeric transcription factor NF-Y binds to CCAAT boxes of genes of glutamine metabolism. We set out to study the role of the regulatory NF-YA subunit in this pathway. We produced U2OS and A549 clones stably overexpressing -OE- the two splicing isoforms of NF-YA. NF-YA OE cells show normal growth and colony formation rates, but they become resistant to cell death upon glutamine deprivation. Increased mRNA and protein expression of the key biosynthetic enzyme GLUL in U2OS entails increased production of endogenous glutamine upon deprivation. The use of GLUL inhibitors dampens the NF-YA-mediated effect. NF-YA OE prevents activation of the pro-apoptotic transcription factor CHOP/DDIT3. Elevated basal levels of SERCA1/2, coding for the molecular target of Thapsigargin, correlate with resistance of NF-YA OE cells to the drug. The work represents a proof-of-principle that elevated levels of NF-YA, as found in some tumor types, helps altering cancer metabolic pathways.
    Keywords:  ER-stress; GLUL; Glutamine deprivation; NF-Y