Integrative Molecular Phenotyping
INTEGRATIVE MOLECULAR
PHENOTYPING
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY
DEPARTMENT OF MEDICAL
BIOCHEMISTRY AND BIOPHYSICS
WHEELOCK LABORATORY

PubMed

PubMed
NCBI: db=pubmed; Term=metabolomics
Updated: 4 min 46 sec ago

Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.

Fri, 19/01/2018 - 13:36
Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry. PLoS One. 2018;13(1):e0191230 Authors: Harada S, Hirayama A, Chan Q, Kurihara A, Fukai K, Iida M, Kato S, Sugiyama D, Kuwabara K, Takeuchi A, Akiyama M, Okamura T, Ebbels TMD, Elliott P, Tomita M, Sato A, Suzuki C, Sugimoto M, Soga T, Takebayashi T Abstract BACKGROUND: Cohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population. METHODS: Metabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid. RESULTS AND CONCLUSIONS: In QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies. PMID: 29346414 [PubMed - in process]

Enhanced Isotopic Ratio Outlier Analysis (IROA) Peak Detection and Identification with Ultra-High Resolution GC-Orbitrap/MS: Potential Application for Investigation of Model Organism Metabolomes.

Fri, 19/01/2018 - 13:36
Enhanced Isotopic Ratio Outlier Analysis (IROA) Peak Detection and Identification with Ultra-High Resolution GC-Orbitrap/MS: Potential Application for Investigation of Model Organism Metabolomes. Metabolites. 2018 Jan 18;8(1): Authors: Qiu Y, Moir RD, Willis IM, Seethapathy S, Biniakewitz RC, Kurland IJ Abstract Identifying non-annotated peaks may have a significant impact on the understanding of biological systems. In silico methodologies have focused on ESI LC/MS/MS for identifying non-annotated MS peaks. In this study, we employed in silico methodology to develop an Isotopic Ratio Outlier Analysis (IROA) workflow using enhanced mass spectrometric data acquired with the ultra-high resolution GC-Orbitrap/MS to determine the identity of non-annotated metabolites. The higher resolution of the GC-Orbitrap/MS, together with its wide dynamic range, resulted in more IROA peak pairs detected, and increased reliability of chemical formulae generation (CFG). IROA uses two different 13C-enriched carbon sources (randomized 95% 12C and 95% 13C) to produce mirror image isotopologue pairs, whose mass difference reveals the carbon chain length (n), which aids in the identification of endogenous metabolites. Accurate m/z, n, and derivatization information are obtained from our GC/MS workflow for unknown metabolite identification, and aids in silico methodologies for identifying isomeric and non-annotated metabolites. We were able to mine more mass spectral information using the same Saccharomyces cerevisiae growth protocol (Qiu et al. Anal. Chem 2016) with the ultra-high resolution GC-Orbitrap/MS, using 10% ammonia in methane as the CI reagent gas. We identified 244 IROA peaks pairs, which significantly increased IROA detection capability compared with our previous report (126 IROA peak pairs using a GC-TOF/MS machine). For 55 selected metabolites identified from matched IROA CI and EI spectra, using the GC-Orbitrap/MS vs. GC-TOF/MS, the average mass deviation for GC-Orbitrap/MS was 1.48 ppm, however, the average mass deviation was 32.2 ppm for the GC-TOF/MS machine. In summary, the higher resolution and wider dynamic range of the GC-Orbitrap/MS enabled more accurate CFG, and the coupling of accurate mass GC/MS IROA methodology with in silico fragmentation has great potential in unknown metabolite identification, with applications for characterizing model organism networks. PMID: 29346327 [PubMed]

A metabolomics study reveals enhanced inhibition and metabolic dysregulation in E. coli induced by Lactobacillus acidophilus fermented black tea extract.

Fri, 19/01/2018 - 13:36
A metabolomics study reveals enhanced inhibition and metabolic dysregulation in E. coli induced by Lactobacillus acidophilus fermented black tea extract. J Agric Food Chem. 2018 Jan 18;: Authors: Yang K, Duley M, Zhu J Abstract This study examined the ability of Lactobacillus acidophilus (LA) to ferment black tea extract (BTE), and the enhancement of Escherichia coli cellular uptake of phenolic compounds when this bacteria was incubated with fermented BTE. The inhibitory effects of BTE to E. coli bacteria, with and w/o fermentation, were compared. Several intracellular phenolic compounds, as well as metabolic profiles of E. coli with and w/o treatments, were also determined using a HPLC-MS/MS-based approach. Our results showed that out of three concentrations from the non-fermented BTE treatment, only the extract from the 25 mg/ml tea leaves solution could inhibit E. coli survival, while LA fermented BTE extract from 5, 10 and 25 mg/mL tea leaves solutions all inhibited E. coli growth significantly. Intracellular concentration of (+)-catechin-3-gallate/ (-)-epicatechin-3-gallate and (+)-catechin / (-)-epicatechin were significantly higher when E. coli was treated with fermented BTE in comparison to non-fermented BTE. Scanning electron microscopy (SEM) images indicated that the intracellular phenolic compounds inhibited E. coli growth by increasing endogenous oxidative stress. Metabolic profiles of E. coli were also investigated to understand their metabolic response when treated with BTE, and significant metabolic changes of E. coli were observed. Metabolic profile data were further analyzed using partial least square-discriminant analysis (PLS-DA) to distinguish the fermented BTE treatment group from the control group and the non-fermented BTE treatment group. The results indicated a large-scale E. coli metabolic dysregulation induced by the fermented BTE. Our findings showed that LA fermentation can be an efficient approach to enhance phenolic inhibition of bacterial cells through increased endogenous oxidative stress and dysregulated metabolic activities. PMID: 29345909 [PubMed - as supplied by publisher]

Application of molecular tools to elucidate the microbiota of seafood.

Fri, 19/01/2018 - 13:36
Related Articles Application of molecular tools to elucidate the microbiota of seafood. J Appl Microbiol. 2018 Jan 18;: Authors: de Almeida Rodrigues P, Ferrari RG, Conte Junior CA Abstract The aim of this review is to present the methodologies currently applied to identify microbiota and pathogens transmitted to humans through seafood consumption, focusing on molecular techniques and pointing out their importance, advantages, disadvantages, and applicability. Knowledge of available techniques allows researchers to identify which technique best fits their expectations. With such discernment, it will be possible to infer which disadvantages will be present and, therefore, not interfering with the final result. Two methodologies can be employed for this purpose, dependent and independent cultures. However, the culture-dependent has certain limitations that can be solved through the independent cultivation techniques, such as PCR, PFGE and NGS, especially through the sequencing of the 16S rRNA region, providing a complete view of microbial diversity. These have revolutionized microbiological knowledge, mainly because they allow for the identification of uncultivable microorganisms, which represent a substantial portion of total microorganisms, making it possible to elucidate not yet described taxa which may display pathogenic potential, besides quantifying microbial communities, microbiota genetics, translated proteins and produced metabolites. In addition, transcriptomic and metabolomics techniques also allow for the evaluation of possible impacts that microbial communities may create in their environment, as well as the determination of potential pathogenicity to humans. This article is protected by copyright. All rights reserved. PMID: 29345036 [PubMed - as supplied by publisher]

Effluent and serum protein N-glycosylation is associated with inflammation and peritoneal membrane transport characteristics in peritoneal dialysis patients.

Fri, 19/01/2018 - 13:36
Related Articles Effluent and serum protein N-glycosylation is associated with inflammation and peritoneal membrane transport characteristics in peritoneal dialysis patients. Sci Rep. 2018 Jan 17;8(1):979 Authors: Ferrantelli E, Farhat K, Ederveen ALH, Reiding KR, Beelen RHJ, van Ittersum FJ, Wuhrer M, Dotz V Abstract Mass spectrometric glycomics was used as an innovative approach to identify biomarkers in serum and dialysate samples from peritoneal dialysis (PD) patients. PD is a life-saving treatment worldwide applied in more than 100,000 patients suffering from chronic kidney disease. PD treatment uses the peritoneum as a natural membrane to exchange waste products from blood to a glucose-based solution. Daily exposure of the peritoneal membrane to these solutions may cause complications such as peritonitis, fibrosis and inflammation which, in the long term, lead to the failure of the treatment. It has been shown in the last years that protein N-glycosylation is related to inflammatory and fibrotic processes. Here, by using a recently developed MALDI-TOF-MS method with linkage-specific sialic acid derivatisation, we showed that alpha2,6-sialylation, especially in triantennary N-glycans from peritoneal effluents, is associated with critical clinical outcomes in a prospective cohort of 94 PD patients. Moreover, we found an association between the levels of presumably immunoglobulin-G-related glycans as well as galactosylation of diantennary glycans with PD-related complications such as peritonitis and loss of peritoneal mesothelial cell mass. The observed glycomic changes point to changes in protein abundance and protein-specific glycosylation, representing candidate functional biomarkers of PD and associated complications. PMID: 29343697 [PubMed - in process]

Uremic Solute-Aryl Hydrocarbon Receptor-Tissue Factor Axis Associates with Thrombosis after Vascular Injury in Humans.

Fri, 19/01/2018 - 13:36
Related Articles Uremic Solute-Aryl Hydrocarbon Receptor-Tissue Factor Axis Associates with Thrombosis after Vascular Injury in Humans. J Am Soc Nephrol. 2018 Jan 17;: Authors: Kolachalama VB, Shashar M, Alousi F, Shivanna S, Rijal K, Belghasem ME, Walker J, Matsuura S, Chang GH, Gibson CM, Dember LM, Francis JM, Ravid K, Chitalia VC Abstract Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis. PMID: 29343519 [PubMed - as supplied by publisher]

Mass-spectrometric profiling of cerebrospinal fluid reveals metabolite biomarkers for CNS involvement in varicella zoster virus reactivation.

Fri, 19/01/2018 - 13:36
Related Articles Mass-spectrometric profiling of cerebrospinal fluid reveals metabolite biomarkers for CNS involvement in varicella zoster virus reactivation. J Neuroinflammation. 2018 Jan 17;15(1):20 Authors: Kuhn M, Sühs KW, Akmatov MK, Klawonn F, Wang J, Skripuletz T, Kaever V, Stangel M, Pessler F Abstract BACKGROUND: Varicella zoster virus (VZV) reactivation spans the spectrum from uncomplicated segmental herpes zoster to life-threatening disseminated CNS infection. Moreover, in the absence of a small animal model for this human pathogen, studies of pathogenesis at the organismal level depend on analysis of human biosamples. Changes in cerebrospinal fluid (CSF) metabolites may reflect critical aspects of host responses and end-organ damage in neuroinfection and neuroinflammation. We therefore applied a targeted metabolomics screen of CSF to three clinically distinct forms of VZV reactivation and infectious and non-infectious disease controls in order to identify biomarkers for CNS involvement in VZV reactivation. METHODS: Metabolite profiles were determined by targeted liquid chromatography-mass spectrometry in CSF from patients with segmental zoster (shingles, n = 14), facial nerve zoster (n = 16), VZV meningitis/encephalitis (n = 15), enteroviral meningitis (n = 10), idiopathic Bell's palsy (n = 11), and normal pressure hydrocephalus (n = 15). RESULTS: Concentrations of 88 metabolites passing quality assessment clearly separated the three VZV reactivation forms from each other and from the non-infected samples. Internal cross-validation identified four metabolites (SM C16:1, glycine, lysoPC a C26:1, PC ae C34:0) that were particularly associated with VZV meningoencephalitis. SM(OH) C14:1 accurately distinguished facial nerve zoster from Bell's palsy. Random forest construction revealed even more accurate classifiers (signatures comprising 2-4 metabolites) for most comparisons. Some of the most accurate biomarkers correlated only weakly with CSF leukocyte count, indicating that they do not merely reflect recruitment of inflammatory cells but, rather, specific pathophysiological mechanisms. Across all samples, only the sum of hexoses and the amino acids arginine, serine, and tryptophan correlated negatively with leukocyte count. Increased expression of the metabolites associated with VZV meningoencephalitis could be linked to processes relating to neuroinflammation/immune activation, neuronal signaling, and cell stress, turnover, and death (e.g., autophagy and apoptosis), suggesting that these metabolites might sense processes relating to end-organ damage. CONCLUSIONS: The results provide proof-of-concept for the value of CSF metabolites as (1) disease-associated signatures suggesting pathophysiological mechanisms, (2) degree and nature of neuroinflammation, and (3) biomarkers for diagnosis and risk stratification of VZV reactivation and, likely, neuroinfections due to other pathogens. TRIAL REGISTRATION: Not applicable (non-interventional study). PMID: 29343258 [PubMed - in process]

Impact of Prolonged Blood Incubation and Extended Serum Storage at Room Temperature on the Human Serum Metabolome.

Fri, 19/01/2018 - 13:36
Related Articles Impact of Prolonged Blood Incubation and Extended Serum Storage at Room Temperature on the Human Serum Metabolome. Metabolites. 2018 Jan 13;8(1): Authors: Kamlage B, Neuber S, Bethan B, González Maldonado S, Wagner-Golbs A, Peter E, Schmitz O, Schatz P Abstract Metabolomics is a powerful technology with broad applications in life science that, like other -omics approaches, requires high-quality samples to achieve reliable results and ensure reproducibility. Therefore, along with quality assurance, methods to assess sample quality regarding pre-analytical confounders are urgently needed. In this study, we analyzed the response of the human serum metabolome to pre-analytical variations comprising prolonged blood incubation and extended serum storage at room temperature by using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) -based metabolomics. We found that the prolonged incubation of blood results in a statistically significant 20% increase and 4% decrease of 225 tested serum metabolites. Extended serum storage affected 21% of the analyzed metabolites (14% increased, 7% decreased). Amino acids and nucleobases showed the highest percentage of changed metabolites in both confounding conditions, whereas lipids were remarkably stable. Interestingly, the amounts of taurine and O-phosphoethanolamine, which have both been discussed as biomarkers for various diseases, were 1.8- and 2.9-fold increased after 6 h of blood incubation. Since we found that both are more stable in ethylenediaminetetraacetic acid (EDTA) blood, EDTA plasma should be the preferred metabolomics matrix. PMID: 29342854 [PubMed]

Germline BRCA1 mutation reprograms breast epithelial cell metabolism towards mitochondrial-dependent biosynthesis: evidence for metformin-based "starvation" strategies in BRCA1 carriers.

Fri, 19/01/2018 - 13:36
Related Articles Germline BRCA1 mutation reprograms breast epithelial cell metabolism towards mitochondrial-dependent biosynthesis: evidence for metformin-based "starvation" strategies in BRCA1 carriers. Oncotarget. 2016 Aug 16;7(33):52974-52992 Authors: Cuyàs E, Fernández-Arroyo S, Alarcón T, Lupu R, Joven J, Menendez JA Abstract We hypothesized that women inheriting one germline mutation of the BRCA1 gene ("one-hit") undergo cell-type-specific metabolic reprogramming that supports the high biosynthetic requirements of breast epithelial cells to progress to a fully malignant phenotype. Targeted metabolomic analysis was performed in isogenic pairs of nontumorigenic human breast epithelial cells in which the knock-in of 185delAG mutation in a single BRCA1 allele leads to genomic instability. Mutant BRCA1 one-hit epithelial cells displayed constitutively enhanced activation of biosynthetic nodes within mitochondria. This metabolic rewiring involved the increased incorporation of glutamine- and glucose-dependent carbon into tricarboxylic acid (TCA) cycle metabolite pools to ultimately generate elevated levels of acetyl-CoA and malonyl-CoA, the major building blocks for lipid biosynthesis. The significant increase of branched-chain amino acids (BCAAs) including the anabolic trigger leucine, which can not only promote protein translation via mTOR but also feed into the TCA cycle via succinyl-CoA, further underscored the anabolic reprogramming of BRCA1 haploinsufficient cells. The anti-diabetic biguanide metformin "reversed" the metabolomic signature and anabolic phenotype of BRCA1 one-hit cells by shutting down mitochondria-driven generation of precursors for lipogenic pathways and reducing the BCAA pool for protein synthesis and TCA fueling. Metformin-induced restriction of mitochondrial biosynthetic capacity was sufficient to impair the tumor-initiating capacity of BRCA1 one-hit cells in mammosphere assays. Metabolic rewiring of the breast epithelium towards increased anabolism might constitute an unanticipated and inherited form of metabolic reprogramming linked to increased risk of oncogenesis in women bearing pathogenic germline BRCA1 mutations. The ability of metformin to constrain the production of mitochondrial-dependent biosynthetic intermediates might open a new avenue for "starvation" chemopreventive strategies in BRCA1 carriers. PMID: 27259235 [PubMed - indexed for MEDLINE]

metabolomics; +22 new citations

Thu, 18/01/2018 - 16:21
22 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2018/01/18PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

metabolomics; +22 new citations

Thu, 18/01/2018 - 13:17
22 new pubmed citations were retrieved for your search. Click on the search hyperlink below to display the complete search results: metabolomics These pubmed results were generated on 2018/01/18PubMed comprises more than millions of citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Impact of genistein on the gut microbiome of humanized mice and its role in breast tumor inhibition.

Thu, 18/01/2018 - 01:02
Related Articles Impact of genistein on the gut microbiome of humanized mice and its role in breast tumor inhibition. PLoS One. 2017;12(12):e0189756 Authors: Paul B, Royston KJ, Li Y, Stoll ML, Skibola CF, Wilson LS, Barnes S, Morrow CD, Tollefsbol TO Abstract Since dietary polyphenols can have beneficial effects in prevention and treatment of cancer, we tested the hypothesis that breast cancer patients' intestinal microbiota is modulated by genistein (GE), an isoflavone found in soy, and that microbial alterations may offset the side effects brought about by chemotherapy. We demonstrated successful humanization of germ-free mice by transplanting fecal samples from breast cancer patients before and after chemotherapy. Mice were then grouped based on chemotherapy status and GE or control diet. We did not find any significant differences between pre-chemotherapy and post-chemotherapy bacterial composition and abundances. Germ-free mice on a GE diet showed differences in microbial composition as compared to mice on control diet. Four weeks after introduction of the customized GE diet, there was distinct clustering of GE-fed mice as compared to the control-fed group. In the gut microbiome of GE-treated humanized mice, there was an increase in abundance of genera Lactococcus and Eubacterium. Phylum Verrucomicrobia showed statistically significant (p = 0.02) differences in abundances between the GE-fed and control-fed groups. There was an increase in bacteria belonging to family Lachnospiraceae and Ruminococcaceae in GE-fed mice. Marked changes were observed in GE catabolism in mice humanized with fecal material from two of three patients' post-chemotherapy with complete disappearance of 4-ethylphenol and 2-(4-hydroxyphenol) propionic acid conjugates. The post-tumor samples did not show any distinct clustering of the gut microbiota between the two diet groups. There was an increase in latency of about 25% for tumor growth of the humanized mice that were on a GE diet as compared to humanized mice on a control diet. The average tumor size for the GE group was significantly decreased compared to the non-GE group. Collectively, our results suggest that the intestinal microbiota becomes altered with a GE diet before induction of tumor. Our findings indicate that GE modulates the microbiome in humanized mice that may contribute to its effects on increasing the latency of breast tumor and reducing tumor growth. PMID: 29267377 [PubMed - in process]

Metabolomic profiling and stable isotope labelling of Trichomonas vaginalis and Tritrichomonas foetus reveal major differences in amino acid metabolism including the production of 2-hydroxyisocaproic acid, cystathionine and S-methylcysteine.

Thu, 18/01/2018 - 01:02
Related Articles Metabolomic profiling and stable isotope labelling of Trichomonas vaginalis and Tritrichomonas foetus reveal major differences in amino acid metabolism including the production of 2-hydroxyisocaproic acid, cystathionine and S-methylcysteine. PLoS One. 2017;12(12):e0189072 Authors: Westrop GD, Wang L, Blackburn GJ, Zhang T, Zheng L, Watson DG, Coombs GH Abstract Trichomonas vaginalis and Tritrichomonas foetus are pathogens that parasitise, respectively, human and bovine urogenital tracts causing disease. Using LC-MS, reference metabolomic profiles were obtained for both species and stable isotope labelling with D-[U-13C6] glucose was used to analyse central carbon metabolism. This facilitated a comparison of the metabolic pathways of T. vaginalis and T. foetus, extending earlier targeted biochemical studies. 43 metabolites, whose identities were confirmed by comparison of their retention times with authentic standards, occurred at more than 3-fold difference in peak intensity between T. vaginalis and T. foetus. 18 metabolites that were removed from or released into the medium during growth also showed more than 3-fold difference between the species. Major differences were observed in cysteine and methionine metabolism in which homocysteine, produced as a bi-product of trans-methylation, is catabolised by methionine γ-lyase in T. vaginalis but converted to cystathionine in T. foetus. Both species synthesise methylthioadenosine by an unusual mechanism, but it is not used as a substrate for methionine recycling. T. vaginalis also produces and exports high levels of S-methylcysteine, whereas only negligible levels were found in T. foetus which maintains significantly higher intracellular levels of cysteine. 13C-labeling confirmed that both cysteine and S-methylcysteine are synthesised by T. vaginalis; S-methylcysteine can be generated by recombinant T. vaginalis cysteine synthase using phosphoserine and methanethiol. T. foetus contained higher levels of ornithine and citrulline than T. vaginalis and exported increased levels of putrescine, suggesting greater flux through the arginine dihydrolase pathway. T. vaginalis produced and exported hydroxy acid derivatives of certain amino acids, particularly 2-hydroxyisocaproic acid derived from leucine, whereas negligible levels of these metabolites occurred in T. foetus. PMID: 29267346 [PubMed - in process]

Major roles for minor bacterial lipids identified by mass spectrometry.

Thu, 18/01/2018 - 01:02
Related Articles Major roles for minor bacterial lipids identified by mass spectrometry. Biochim Biophys Acta. 2017 11;1862(11):1319-1324 Authors: Garrett TA Abstract Mass spectrometry of lipids, especially those isolated from bacteria, has ballooned over the past two decades, affirming in the process the complexity of the lipidome. With this has come the identification of new and interesting lipid structures. Here is an overview of several novel lipids, from both Gram-negative and Gram-positive bacteria with roles in health and disease, whose structural identification was facilitated using mass spectrometry. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. PMID: 27760388 [PubMed - in process]

High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells.

Thu, 18/01/2018 - 01:02
Related Articles High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells. Methods Mol Biol. 2017;1503:185-196 Authors: Holst S, van Pelt GW, Mesker WE, Tollenaar RA, Belo AI, van Die I, Rombouts Y, Wuhrer M Abstract The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile. PMID: 27743367 [PubMed - in process]

Site-Specific N- and O-Glycopeptide Analysis Using an Integrated C18-PGC-LC-ESI-QTOF-MS/MS Approach.

Thu, 18/01/2018 - 01:02
Related Articles Site-Specific N- and O-Glycopeptide Analysis Using an Integrated C18-PGC-LC-ESI-QTOF-MS/MS Approach. Methods Mol Biol. 2017;1503:109-119 Authors: Stavenhagen K, Hinneburg H, Kolarich D, Wuhrer M Abstract The vast heterogeneity of protein glycosylation, even of a single glycoprotein with only one glycosylation site, can give rise to a set of macromolecules with different physicochemical properties. Thus, the use of orthogonal approaches for comprehensive characterization of glycoproteins is a key requirement. This chapter describes a universal workflow for site-specific N- and O-glycopeptide analysis. In a first step glycoproteins are treated with Pronase to generate glycopeptides containing small peptide sequences for enhanced glycosylation site assignment and characterization. These glycopeptides are then separated and detected using an integrated C18-porous graphitized carbon-liquid chromatography (PGC-LC) setup online coupled to a high-resolution electrospray ionization (ESI)-quadrupole time-of-flight (QTOF)-mass spectrometer operated in a combined higher- and lower-energy CID (stepping-energy CID) mode. The LC-setup allows retention of more hydrophobic glycopeptides on C18 followed by subsequent capturing of C18-unbound (glyco)peptides by a downstream placed PGC stationary phase. Glycopeptides eluted from both columns are then analyzed within a single analysis in a combined data acquisition mode. Stepping-energy CID results in B- and Y-ion fragments originating from the glycan moiety as well as b- and y-ions derived from the peptide part. This allows simultaneous site-specific identification of the glycan and peptide sequence of a glycoprotein. PMID: 27743362 [PubMed - in process]

Sialic Acid Derivatization for the Rapid Subclass- and Sialic Acid Linkage-Specific MALDI-TOF-MS Analysis of IgG Fc-Glycopeptides.

Thu, 18/01/2018 - 01:02
Related Articles Sialic Acid Derivatization for the Rapid Subclass- and Sialic Acid Linkage-Specific MALDI-TOF-MS Analysis of IgG Fc-Glycopeptides. Methods Mol Biol. 2017;1503:49-62 Authors: de Haan N, Reiding KR, Wuhrer M Abstract Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is a highly suitable method for the rapid analysis of IgG glycopeptides, providing a wealth of structural information. A limitation of this approach is that it generates a bias when analyzing sialylated species due to the labile nature of sialic acid glycosidic linkages. One way to overcome this problem is by chemical derivatization of the sialic acids. The method presented here results in both the stabilization of the sialic acids, as well as the differentiation of α2,3- and α2,6-linked sialic acids by mass. Described in this chapter are the isolation of IgG from plasma or serum, tryptic digestion of the samples, derivatization, and finally MALDI-TOF-MS measurement and data analysis. PMID: 27743358 [PubMed - in process]

High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS.

Thu, 18/01/2018 - 01:02
Related Articles High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS. Methods Mol Biol. 2017;1503:31-47 Authors: Falck D, Jansen BC, de Haan N, Wuhrer M Abstract This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs. PMID: 27743357 [PubMed - in process]

Development and application of a comprehensive lipidomic analysis to investigate Tripterygium wilfordii-induced liver injury.

Thu, 18/01/2018 - 01:02
Related Articles Development and application of a comprehensive lipidomic analysis to investigate Tripterygium wilfordii-induced liver injury. Anal Bioanal Chem. 2016 Jun;408(16):4341-55 Authors: Xie T, Zhou X, Wang S, Lu Y, Zhu H, Kang A, Deng H, Xu J, Shen C, Di L, Shan J Abstract Lipid metabolic pathways play pivotal roles in liver function, and disturbances of these pathways are associated with various diseases. Thus, comprehensive characterization and measurement of lipid metabolites are essential to deciphering the contributions of lipid network metabolism to diseases or its responses to drug intervention. Here, we report an integrated lipidomic analysis for the comprehensive detection of lipid metabolites. To facilitate the characterization of untargeted lipids through fragmentation analysis, nine formulas were proposed to identify the fatty acid composition of lipids from complex MS (n) spectrum information. By these formulas, the co-eluted isomeric compounds could be distinguished. In total, 250 lipids were detected and characterized, including diacylglycerols, triacylglycerols, glycerophosphoethanolamines, glycerophosphocholines, glycerophosphoserines, glycerophosphoglycerols, glycerophosphoinositols, cardiolipins, ceramides, and sphingomyelins. Integrated with the targeted lipidomics, a total of 27 inflammatory oxylipins were also measured. To evaluate the aberrant lipid metabolism involved in liver injury induced by Tripterygium wilfordii, lipid network metabolism was further investigated. Results indicated that energy lipid modification, membrane remodeling, potential signaling lipid alterations, and abnormal inflammation response were associated with injury. Because of the important roles of lipids in liver metabolism, this new method is expected to be useful in analyzing other lipid metabolism diseases. PMID: 27086014 [PubMed - in process]

Editorial: Managing Strategies for Diverse Diseases: Challenges from Bench to Bedside Translation in Successful Drug Discovery and Development (Part B).

Thu, 18/01/2018 - 01:02
Related Articles Editorial: Managing Strategies for Diverse Diseases: Challenges from Bench to Bedside Translation in Successful Drug Discovery and Development (Part B). Curr Pharm Des. 2016;22(20):2923-5 Authors: Kamal MA, Greig NH PMID: 27063488 [PubMed - in process]

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