bims-oximas Biomed News
on Oxidative stress and mass spectrometry
Issue of 2020‒05‒03
twenty-two papers selected by
Alpesh Thakker
University of the Highlands and Islands


  1. Nat Commun. 2020 Apr 28. 11(1): 2057
    Peng B, Kopczynski D, Pratt BS, Ejsing CS, Burla B, Hermansson M, Benke PI, Tan SH, Chan MY, Torta F, Schwudke D, Meckelmann SW, Coman C, Schmitz OJ, MacLean B, Manke MC, Borst O, Wenk MR, Hoffmann N, Ahrends R.
      Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biological conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets.
    DOI:  https://doi.org/10.1038/s41467-020-15960-z
  2. ACS Synth Biol. 2020 Apr 29.
    Riaz MR, Preston GM, Mithani A.
      Comparative and evolutionary analyses of metabolic networks have a wide range of applications, ranging from research into metabolic evolution through to practical applications in drug development, synthetic biology, and biodegradation. We present MAPPS: Metabolic network Analysis and Pathway Prediction Server (https://mapps.lums.edu.pk), a web-based tool to study functions and evolution of metabolic networks using traditional and 'omics data sets. MAPPS provides diverse functionalities including an interactive interface, graphical visualization of results, pathway prediction and network comparison, identification of potential drug targets, in silico metabolic engineering, host-microbe interactions, and ancestral network building. Importantly, MAPPS also allows users to upload custom data, thus enabling metabolic analyses on draft and custom genomes, and has an 'omics pipeline to filter pathway results, making it relevant in today's postgenomic era.
    Keywords:  host−microbe interaction; in silico metabolic engineering; metabolic evolution; metabolic network; network comparison; pathway prediction; ‘omics pipeline
    DOI:  https://doi.org/10.1021/acssynbio.9b00397
  3. Glycobiology. 2020 Apr 27. pii: cwaa038. [Epub ahead of print]
    Wong M, Xu G, Barboza M, Maezawa I, Jin LW, Zivkovic A, Lebrilla CB.
      Saccharides in our diet are major sources of carbon for the formation of biomass such as proteins, lipids, nucleic acids, and glycans. Among the dietary monosaccharides, glucose occupies a central role in metabolism, but human blood contains regulated levels of other monosaccharides as well. Their influence on metabolism and how they are utilized has not been explored thoroughly. Applying metabolic flux analysis on glycan synthesis can reveal the pathways that supply glycosylation precursors and provide a snapshot of the metabolic state of the cell. In this study we traced the incorporation of six 13C uniformly-labeled monosaccharides in the N-glycans, O-glycans, and glycosphingolipids of both pluripotent and neural NTERA-2 cells. We gathered detailed isotopologue data for hundreds of glycoconjugates using mass spectrometry methods. The contributions of de novo synthesis and direct incorporation pathways for glucose, mannose, fructose, galactose, N-acetylglucosamine, and fucose were determined based on their isotope incorporation. Co-feeding studies revealed that fructose incorporation is drastically decreased by the presence of glucose, while mannose and galactose were much less affected. Furthermore, increased sialylation slowed down the turnover of glycans but fucosylation attenuated this effect. Our results demonstrated that exogenous monosaccharide utilization can vary markedly depending on the cell differentiation state and monosaccharide availability, and that the incorporation of carbons can also differ among different glycan structures. We contend that the analysis of metabolic isotope labeling of glycans can yield new insights about cell metabolism.
    Keywords:  glycomics; mass spectrometry; metabolic flux analysis; neuron; stable isotope labeling
    DOI:  https://doi.org/10.1093/glycob/cwaa038
  4. Free Radic Res. 2020 Apr 26. 1-23
    Saito K, Matsuoka Y, Yamada KI.
      Lipid peroxidation (LPO) is reported to be involved in the pathogenesis of several oxidative diseases, and several therapeutic approaches using antioxidants have been proposed. LPO is thought to progress via a complicated series of multistep reactions suggesting that the activity of each antioxidant may be different, and depends on the reacting molecules. Hence, in this study, we evaluated the inhibitory mechanisms of several antioxidants toward arachidonic acid (AA) peroxidation induced by the azo initiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) or a lipid hydroperoxide, hydroperoxyoctadecadienoic acid (HpODE)/hemin. Edaravone, ferrostain-1, TEMPO and trolox effectively inhibited the production of malondialdehyde (MDA) and several oxidized AAs generated in the AAPH-induced LPO because of their scavenging ability toward lipid peroxyl radicals. In contrast, ebselen and ferrostatin-1 showed strong antioxidative activity in the HpODE/hemin-induced peroxidation. Under this condition, ebselen and ferrostatin-1 were thought to reduce HpODE and its derived alkoxyl radicals to the corresponding lipid alcohols. In conclusion, we found that each antioxidant had different antioxidative activities that prevented the progression of LPO. We expect that these findings will contribute to the design of novel therapeutic strategies using an appropriate antioxidant targeted to each step of the development of oxidative stress diseases.
    Keywords:  antioxidant; lipid hydroperoxide; lipid peroxidation; lipid radical
    DOI:  https://doi.org/10.1080/10715762.2020.1761020
  5. Anal Bioanal Chem. 2020 Apr 29.
    Xu M, Legradi J, Leonards P.
      In this study, both conventional one-dimensional liquid chromatography (1DLC) and comprehensive two-dimensional liquid chromatography (2DLC) coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF MS) were used for full-scale lipid characterization of lipid extracts from zebrafish embryos. We investigated the influence on annotated lipids and different separation mechanisms (HILIC, C18, and PFP), and their different orders arranged in the first and the second dimensions. As a result, the number of lipid species annotated by conventional one-dimensional LC-MS was between 212 and 448. In contrast, the number of individual lipids species annotated by C18×HILIC, HILIC×C18, and HILIC×PFP were 1784, 1059, and 1123, respectively. Therefore, it was evident that the performance of comprehensive 2DLC, especially the C18×HILIC method, considerably exceeded 1DLC. Interestingly, a comparison of the HILIC×C18 and C18×HILIC approaches showed, under the optimized conditions, similar orthogonality, but the effective separation power of the C18×HILIC was much higher. A comparison of the HILIC×C18 and the HILIC×PFP methods demonstrated that the HILIC×PFP separation had superior orthogonality with a small increase on its effective peak capacity, indicating that the HILIC×PFP combination maybe a promising platform for untargeted lipidomics in complex samples. Finally, from the comprehensive lipid profiling respective, the C18×HILIC was selected for further studies.
    Keywords:  Comprehensive two-dimensional liquid chromatography; Conventional one-dimensional liquid chromatography; Untargeted lipidomics; Zebrafish
    DOI:  https://doi.org/10.1007/s00216-020-02661-1
  6. J Lipid Res. 2020 Apr 27. pii: jlr.D120000809. [Epub ahead of print]
    Rohrbach TD, Boyd AE, Grizzard PJ, Spiegel S, Allegood J, Lima S.
      Mass spectrometry (MS) assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of optimal cutting temperature compound (OCT) used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT also interferes with protein quantification assays, and that the presence of OCT impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT from OCT-embedded tissues. Our results indicate that removal of OCT from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer, and to determine the long-term stability of sphingolipids in OCT-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT-cryopreserved normal lung tissues. This validated sphingolipidomic OCT-removal protocol should be a valuable addition to the lipid biologist's toolbox.
    Keywords:  Cancer; Ceramides; Lipidomics; Mass spectrometry; OCT; Sphingolipids; biorepository; lung adenocarcinoma; lung squamous cell carcinoma; non-small cell lung cancer
    DOI:  https://doi.org/10.1194/jlr.D120000809
  7. Anal Chim Acta. 2020 May 22. pii: S0003-2670(20)30226-9. [Epub ahead of print]1112 34-45
    Richardson LT, Brantley MR, Solouki T.
      Untargeted mass spectrometry (MS) workflows are more suitable than targeted workflows for high throughput characterization of complex biological samples. However, analysis workflows for untargeted methods are inadequate for characterization of complex samples that contain multiple classes of compounds as each chemical class might require a different type of data processing approach. To increase the feasibility of analyzing MS data for multi-class/component complex mixtures (i.e., mixtures containing more than one major class of biomolecules), we developed a neural network-based approach for classification of MS data. In our in silico fractionation (iSF) approach, we utilize a neural decision tree to sequentially classify biomolecules based on their MS-detected isotopic patterns. In the presented demonstration, the neural decision tree consisted of two supervised binary classifiers to positively classify polypeptides and lipids, respectively, and a third supervised network was trained to classify lipids into the eight main sub-categories of lipids. The two binary classifiers assigned polypeptide and lipid experimental components with 100% sensitivity and 100% specificity; however, the 8-target classifier assigned lipids into their respective subclasses with 95% sensitivity and 99% specificity. Here, we discuss important relationships between class-specific chemical properties and MS isotopic envelopes that enable analyte classification. Moreover, we evaluate the performance characteristics of the utilized networks.
    Keywords:  Chemometrics; Feedforward neural network; Isotopic envelope; Mass spectrometry; Neural decision tree
    DOI:  https://doi.org/10.1016/j.aca.2020.02.036
  8. Electrophoresis. 2020 Apr 26.
    Sándor V, Berkics BV, Kilár A, Kocsis B, Kilár F, Dörnyei Á.
      Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min, in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications. This article is protected by copyright. All rights reserved.
    Keywords:  acylation isomerism; lipid A; nonaqueous capillary electrophoresis-tandem mass spectrometry; normal and reverse polarity; phosphorylation isomerism
    DOI:  https://doi.org/10.1002/elps.201900251
  9. J Chromatogr A. 2020 Apr 20. pii: S0021-9673(20)30296-X. [Epub ahead of print] 461075
    Ferri M, Bäurer S, Carotti A, Wolter M, Alshaar B, Theiner J, Ikegami T, West C, Lämmerhofer M.
      The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative ζ-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted ζ-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations.
    Keywords:  Mixed-mode chromatography; hydrophilic interaction chromatography (HILIC); mixed-mode anion-exchangers; mixed-mode cation-exchangers; molecular dynamics simulation; zwitterionic mixed-mode ion-exchangers
    DOI:  https://doi.org/10.1016/j.chroma.2020.461075
  10. Metabolites. 2020 Apr 28. pii: E175. [Epub ahead of print]10(5):
    Tsamesidis I, Egwu CO, Pério P, Augereau JM, Benoit-Vical F, Reybier K.
      Red blood cells are constantly exposed to reactive species under physiological or pathological conditions or during administration of xenobiotics. Regardless of the source, its accurate quantification is paramount in the area of theragnostics, which had been elusive up until now. Even if there are a lot of approaches to evaluate the oxidative stress, very sensitive methods are missing for the blood system. We therefore sought to apply a highly sensitive approach, by liquid chromatography coupled to mass spectrometry (UPLC-MS), for the quantification of reactive species such as superoxide radical and hydrogen peroxide using dihydroethidium (DHE) and coumarin boronic acid (CBA) probes respectively through the detection of 2-hydroxyethidium (2OH-E+) and 7-hydroxycoumarin (COH). The use of the high-resolution mass spectrometry associated to UPLC ensured a selective detection of superoxide and hydrogen peroxide in the blood system under diverse conditions such as oxidized red blood cells (RBCs), untreated and treated parasitized RBCs. Moreover, this technique allowed the determination of reactive species in human plasma. This protocol provides a huge opportunity for in-depth study of several pathological conditions vis-a-vis their treatment in modern medicine.
    Keywords:  Plasmodium falciparum; human plasma; hydrogen peroxide species; liquid-chromatography; mass spectrometry; microvesicles; red blood cells; superoxide radicals
    DOI:  https://doi.org/10.3390/metabo10050175
  11. J Lipid Res. 2020 Apr 29. pii: jlr.RA119000516. [Epub ahead of print]
    Grabner GF, Fawzy N, Schreiber R, Pusch LM, Bulfon D, Koefeler H, Eichmann TO, Lass A, Schweiger M, Marsche G, Schoiswohl G, Taschler U, Zimmermann R.
      Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endo-lysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and  parenchymal and non-parenchymal cells. Tissue samples were obtained from fed, fasted, two-hours refed, and insulin-treated mice, as well as from mice housed at  5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles, which were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue-and cell type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP possesses a role in the functional adaption to nutrient starvation and ambient temperature.
    Keywords:  Adipose tissue; Insulin; Lipid metabolism; Liver; Pancreas; Phospholipids; bis(monoacylglycero)phosphate (BMP); body temperature; lysosome; nutritional state
    DOI:  https://doi.org/10.1194/jlr.RA119000516
  12. Nat Rev Mol Cell Biol. 2020 Apr 29.
    Levental I.
      
    DOI:  https://doi.org/10.1038/s41580-020-0252-x
  13. Anal Bioanal Chem. 2020 Apr 30.
    Bedia C, Sierra À, Tauler R.
      Current histology techniques, such as tissue staining or histochemistry protocols, provide very limited chemical information about the tissues. Chemical imaging technologies such as infrared, Raman, and mass spectrometry imaging, are powerful analytical techniques with a huge potential in describing the chemical composition of sample surfaces. In this work, three images of the same tissue slice using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, infrared microspectroscopy, and an RGB picture from a conventional hematoxylin/eosin (H/E) staining are simultaneously analyzed. These fused images were analyzed by multivariate curve resolution-alternating least squares (MCR-ALS), which provided, for each component, its distribution within the tissue surface, its IR spectrum fingerprint, its characteristic mass values, and the contribution of the RGB channels of the H/E staining. Compared with the individual analysis of each of the images alone, the fusion of the three images showed the relationship between the different types of chemical/biological information and enabled a better interpretation of the tissue under study. In addition, the least-squares projection of the MCR-ALS resolved spectra of components at low spatial resolution onto the IR and RBG images at high spatial resolution, provided a better delimitation of the sample constituents on the image, giving a more precise description of their distribution on the investigated tissue. The application of this procedure can be of interest in different research areas in which a good description of the spatial distribution of the chemical constituents of the samples is needed, such as in biomedicine, food, or environmental research.
    Keywords:  Histology; Image data fusion; Infrared imaging; MCR-ALS; Mass spectrometry imaging; Multimodal chemical imaging
    DOI:  https://doi.org/10.1007/s00216-020-02595-8
  14. Cell Biol Toxicol. 2020 Apr 28.
    Zhang L, Lv J, Chen C, Wang X.
      Lung cancer is a heterogeneous and complex disease with the highest incidence and mortality rate. The present study aims at defining the lung cancer phenome specificity of lipidomic profiles, screening target lipid-dependent transcriptional alternations, identifying target lipid-associated target genes, and exploring molecular mechanisms. Lung cancer-specific and lung cancer subtype-specific target lipids palmitic acid (C16:0) and stearic acid (C18:0) were found as target lipids by integrating clinical phenomics, lipidomics, and transcriptomics and exhibited antiproliferative effects in sensitive cells. The metabolism-associated gene ACSL5 or inflammation-associated gene CCL3 was identified in lung adenocarcinoma or small lung cancer cells, respectively. C16:0 or C18:0 could upregulate ACSL5 or CSF2 expression in a time- and dose-dependent pattern, and the deletion of both genes led to the insensitivity of cells. Target lipids increased the expression of PDK4 gene in different patterns and inhibited cell proliferation through alterations of intracellular energy. Thus, our data provide a new strategy to investigate the trans-points between clinical and phenomics and lipidomics and target lipid-associated molecular mechanisms to benefit from the discovery of new therapies.
    Keywords:  ACSL5; CSF2; Clinical phenome; Lipidomics; Lung cancer
    DOI:  https://doi.org/10.1007/s10565-020-09520-w
  15. Int J Mol Sci. 2020 Apr 27. pii: E3074. [Epub ahead of print]21(9):
    Cas MD, Roda G, Li F, Secundo F.
      Lipids are apolar small molecules known not only as components of cell membranes but also, in recent literature, as modulators of different biological functions. Herein, we focused on the bioactive lipids that can influence the immune responses and inflammatory processes regulating vascular hyperreactivity, pain, leukocyte trafficking, and clearance. In the case of excessive pro-inflammatory lipid activity, these lipids also contribute to the transition from acute to chronic inflammation. Based on their biochemical function, these lipids can be divided into different families, including eicosanoids, specialized pro-resolving mediators, lysoglycerophospholipids, sphingolipids, and endocannabinoids. These bioactive lipids are involved in all phases of the inflammatory process and the pathophysiology of different chronic autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, type-1 diabetes, and systemic lupus erythematosus.
    Keywords:  endocannabinoids; inflammation; lipids; lysoglycerophospholipids; sphingolipids
    DOI:  https://doi.org/10.3390/ijms21093074
  16. Prog Lipid Res. 2020 Apr 24. pii: S0163-7827(20)30010-2. [Epub ahead of print] 101030
    Ekroos K, Lavrynenko O, Titz B, Pater C, Hoeng J, Ivanov NV.
      For many diseases, there is an unmet need for new or better biomarkers for improved disease risk assessment and monitoring, as available markers lack sufficient specificity. Lipids are drawing major interest as potential candidates for filling these gaps. This has recently been demonstrated by the identification of selective ceramides for prediction of cardiovascular mortality, enabling improved risk assessment of cardiovascular disease compared with conventional clinical markers. In this review, we discuss current lipid biomarker findings and the possible connection between cardiovascular disease, chronic obstructive pulmonary disease, and aging. Moreover, we discuss how to overcome the current roadblocks facing lipid biomarker research. We stress the need for improved quantification, standardization of methodologies, and establishment of initial reference values to allow for an efficient transfer path of research findings into the clinical landscape, and, ultimately, to put newly identified biomarkers into practical use.
    Keywords:  Aging; Biomarkers; Cardiovascular disease; Chronic obstructive pulmonary disease; Lipidomics
    DOI:  https://doi.org/10.1016/j.plipres.2020.101030
  17. J Proteome Res. 2020 Apr 30.
    Pavlaki A, Begou O, Deda O, Farmaki E, Dotis J, Gika H, Taparkou A, Raikos N, Papachristou F, Theodoridis G, Printza N.
      Ureteropelvic junction obstruction (UPJO) constitutes the predominant cause of obstructive nephropathy in both neonates and infants, with fundamental questions regarding its mechanism, assessment and treatment that remain still unanswered. The aim of this study was to elucidate potential differences through serum metabolic profiling of surgical cases of infants with UPJO compared to both non-surgical cases and healthy age matched controls. Early diagnosis of renal dysfunction in this cohort based on highlighted biomarkers was the ultimate goal. Thus, serum samples were collected from 20 patients preoperatively, 19 patients with mild stenosis treated conservatively, and 17 healthy controls. All samples were subjected to targeted metabolomics analysis by Hydrophilic Interaction Liquid Chromatography coupled to mass spectrometry (HILIC LC-MS/MS). Both univariate and multivariate statistical analysis were performed. PCA and OPLS-DA score plots showed that the studied groups differed significantly, with a panel of metabolites, including creatinine, L-tryptophan, choline and L-aspartate, distinguishing patients who required surgery from those followed by systematical monitoring as well as from healthy controls, showing high performance as indicators of UPJO disease. The mass spectrometry data is available at the Center for Computational Mass Spectrometry (ccms) website, where it has been assigned as MassIVE MSV000085063 (ftp://massive.ucsd.edu/MSV000085063/). The data can be accessed directly via it's project doi:10.25345/C5HX3M.
    DOI:  https://doi.org/10.1021/acs.jproteome.9b00855
  18. Rapid Commun Mass Spectrom. 2020 Apr 27. e8818
    Wolters C, Flandinet L, He C, Isa J, Orthous-Daunay FR, Thissen R, Hörst S, Vuitton V.
      RATIONALE: Acquisition quality in analytical science is key to obtaining optimal data from a sample. In very high-resolution mass spectrometry, quality is driven by the optimization of multiple parameters, including the use of scans and micro-scans (or transients) for performing a Fourier transformation.METHODS: 39 mass spectra of a single synthesized complex sample were acquired using various numbers of scan and micro-scan determined through a simple experimental design. An electrospray ion source coupled with an LTQ-Orbitrap-XL mass spectrometer was used and acquisition was performed using a single mass range. All the resulting spectra were treated in the same way to enable comparisons of assigned stoichiometric formulae between acquisitions.
    RESULTS: Converting the number of scans into micro-scans enhances signal quality by lowering noise and reducing artifacts. This modification also increases the number of attributed stoichiometric formulae for an equivalent acquisition time, giving access to a larger molecular diversity for the analyzed complex sample.
    CONCLUSION: For complex samples, the use of long acquisition times leads to optimal data quality, and the use of micro-scans instead of scans-only maximizes the number of attributed stoichiometric formulae.
    DOI:  https://doi.org/10.1002/rcm.8818
  19. Metabolomics. 2020 Apr 25. 16(5): 62
    Erland LAE, Turi CE, Saxena PK, Murch SJ.
      INTRODUCTION: Plants respond to changes in their environments through hormonal activation of a physiological cascade that redirects metabolic resources and growth. In filberts (Corylus sp.), chelated iron promotes the growth of new shoots but the mechanism(s) are not understood.OBJECTIVES: To use untargeted metabolomics and hormonomics approaches to generate novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro.
    METHODS: Data were generated using previously optimized standardized untargeted metabolomics protocols with time of flight mass spectrometry. Multivariate statistical tools (principal component and partial least squares discriminant analysis) did not detect significant differences. Discovery tools Significance Analysis of Microarrays (SAM), multiple linear regression analysis, Bayesian analysis, logical algorithms, machine learning, synthetic biotransformations, targeted hormonomics, and online resources including MetaboAnalyst were used.
    RESULTS: Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose dependent decreases were found in polyphenol metabolism, specifically ellagic acid and its methylated derivative 3,4,3'-tri-O-methylellagic acid. Hormonomics analysis revealed significant differences in phytohormones and their conjugates. FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid, jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated regenerating tissues while the related metabolite melatonin was increased. Eight phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA indicating that metabolism to sequester, deactivate and metabolize 5HT was induced by Fe(III). Tryptophan was metabolized through kynurenine but not anthranilate.
    CONCLUSION: Seven novel hypotheses were generated to guide future studies to understand the regulatory control(s) of shoot organogenesis.
    Keywords:  Corylus; Fe-EDDHA; Indoleamine; Kynurenine; Metabolomics; Micropropagation
    DOI:  https://doi.org/10.1007/s11306-020-01684-0
  20. PLoS One. 2020 ;15(4): e0232283
    Nakano D, Kawaguchi T, Iwamoto H, Hayakawa M, Koga H, Torimura T.
      AIM: Metabolic reprograming is crucial in the proliferation of hepatocellular carcinoma (HCC). Canagliflozin (CANA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, affects various metabolisms. We investigated the effects of CANA on proliferation and metabolic reprograming of HCC cell lines using multi-omics analysis of metabolomics and absolute quantification proteomics (iMPAQT).METHODS: The cells were counted 72 hours after treatment with CANA (10 μM; n = 5) or dimethyl sulfoxide (control [CON]; n = 5) in Hep3B and Huh7 cells. In Hep3B cells, metabolomics and iMPAQT were used to evaluate the levels of metabolites and metabolic enzymes in the CANA and CON groups (each n = 5) 48 hours after treatment.
    RESULTS: Seventy-two hours after treatment, the number of cells in the CANA group was significantly decreased compared to that in the CON group in Hep3B and Huh7 cells. On multi-omics analysis, there was a significant difference in the levels of 85 metabolites and 68 metabolic enzymes between the CANA and CON groups. For instance, CANA significantly downregulated ATP synthase F1 subunit alpha, a mitochondrial electron transport system protein (CON 297.28±20.63 vs. CANA 251.83±22.83 fmol/10 μg protein; P = 0.0183). CANA also significantly upregulated 3-hydroxybutyrate, a beta-oxidation metabolite (CON 530±14 vs. CANA 854±68 arbitrary units; P<0.001). Moreover, CANA significantly downregulated nucleoside diphosphate kinase 1 (CON 110.30±11.37 vs. CANA 89.14±8.39 fmol/10 μg protein; P = 0.0172).
    CONCLUSIONS: We found that CANA suppressed the proliferation of HCC cells through alterations in mitochondrial oxidative phosphorylation metabolism, fatty acid metabolism, and purine and pyrimidine metabolism. Thus, CANA may suppress the proliferation of HCC by regulating metabolic reprograming.
    DOI:  https://doi.org/10.1371/journal.pone.0232283
  21. Metabolites. 2020 Apr 24. pii: E168. [Epub ahead of print]10(4):
    Ioannou GN, Nagana Gowda GA, Djukovic D, Raftery D.
      Nonalcoholic fatty liver disease (NAFLD) is categorized based on histological severity into nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH). We used a multiplatform metabolomics approach to identify metabolite markers and metabolic pathways that distinguish NAFL from early NASH and advanced NASH. We analyzed fasting serum samples from 57 prospectively-recruited patients with histologically-proven NAFLD, including 12 with NAFL, 31 with early NASH and 14 with advanced NASH. Metabolite profiling was performed using a combination of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy analyzed with multivariate statistical and pathway analysis tools. We targeted 237 metabolites of which 158 were quantified. Multivariate analysis uncovered metabolite profile clusters for patients with NAFL, early NASH, and advanced NASH. Also, multiple individual metabolites were associated with histological severity, most notably spermidine which was more than 2-fold lower in advanced fibrosis vs. early fibrosis, in advanced NASH vs. NAFL and in advanced NASH vs. early NASH, suggesting that spermidine exercises a protective effect against development of fibrosing NASH. Furthermore, the results also showed metabolic pathway perturbations between early-NASH and advanced-NASH. In conclusion, using a combination of two reliable analytical platforms (LC-MS and NMR spectroscopy) we identified individual metabolites, metabolite clusters and metabolic pathways that were significantly different between NAFL, early-NASH, and advanced-NASH. These differences provide mechanistic insights as well as potentially important metabolic biomarker candidates that may noninvasively distinguish patients with NAFL, early-NASH, and advanced-NASH. The associations of spermidine levels with less advanced histology merit further assessment of the potential protective effects of spermidine in NAFLD.
    Keywords:  liquid chromatography-mass spectrometry; metabolic pathway; nonalcoholic fatty liver; nonalcoholic steatohepatitis; nuclear magnetic resonance spectroscopy
    DOI:  https://doi.org/10.3390/metabo10040168
  22. Metabolomics. 2020 May 01. 16(5): 64
    Bliziotis NG, Engelke UFH, Aspers RLEG, Engel J, Deinum J, Timmers HJLM, Wevers RA, Kluijtmans LAJ.
      INTRODUCTION: When analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr-Purcell-Meiboom-Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites.OBJECTIVES: Our aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples.
    METHODS: We compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares-Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated.
    RESULTS: We found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation (< 30%) in replicate samples.
    CONCLUSION: Overall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.
    Keywords:  Classification; High-throughput; LED; Large scale; Metabolomics; NMR
    DOI:  https://doi.org/10.1007/s11306-020-01686-y