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MID Max: LC-MS/MS Method for Measuring the Precursor and Product Mass Isotopomer Distributions of Metabolic Intermediates and Cofactors for Metabolic Flux Analysis Applications.
McCloskey D, Young JD, Xu S, Palsson BO, Feist AM
(2016) Anal Chem 88: 1362-70
MeSH Terms: Adenosine Triphosphate, Carbohydrate Metabolism, Chromatography, High Pressure Liquid, Escherichia coli, Isotope Labeling, Metabolic Flux Analysis, Molecular Structure, Tandem Mass Spectrometry
Show Abstract · Added March 31, 2016
The analytical challenges to acquire accurate isotopic data of intracellular metabolic intermediates for stationary, nonstationary, and dynamic metabolic flux analysis (MFA) are numerous. This work presents MID Max, a novel LC-MS/MS workflow, acquisition, and isotopomer deconvolution method for MFA that takes advantage of additional scan types that maximizes the number of mass isotopomer distributions (MIDs) that can be acquired in a given experiment. The analytical method was found to measure the MIDs of 97 metabolites, corresponding to 74 unique metabolite-fragment pairs (32 precursor spectra and 42 product spectra) with accuracy and precision. The compounds measured included metabolic intermediates in central carbohydrate metabolism and cofactors of peripheral metabolism (e.g., ATP). Using only a subset of the acquired MIDs, the method was found to improve the precision of flux estimations and number of resolved exchange fluxes for wild-type E. coli compared to traditional methods and previously published data sets.
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8 MeSH Terms
Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions.
Sarkes DA, Rameh LE
(2016) Methods Mol Biol 1376: 213-27
MeSH Terms: Cell Fractionation, Cell Membrane, Chromatography, High Pressure Liquid, Phosphatidylinositols, Staining and Labeling, Subcellular Fractions, Tritium
Show Abstract · Added November 26, 2018
Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651-657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of the relative phosphoinositide composition of each membrane fraction or organelle and determination of the microenvironment in which each species is enriched. Here, we describe the steps to separate and quantify total or localized phosphoinositides from cultured cells.
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Proteomic Analysis of Macular Fluid Associated With Advanced Glaucomatous Excavation.
Patel S, Ling J, Kim SJ, Schey KL, Rose K, Kuchtey RW
(2016) JAMA Ophthalmol 134: 108-10
MeSH Terms: Chromatography, High Pressure Liquid, Diathermy, Endotamponade, Eye Proteins, Fluorescein Angiography, Humans, Low Tension Glaucoma, Proteomics, Retinoschisis, Subretinal Fluid, Tandem Mass Spectrometry, Tomography, Optical Coherence, Vitrectomy, Vitreous Body
Added February 15, 2016
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14 MeSH Terms
Phase Ib Randomized, Double-Blinded, Placebo-Controlled, Dose Escalation Study of Polyphenon E in Patients with Barrett's Esophagus.
Joe AK, Schnoll-Sussman F, Bresalier RS, Abrams JA, Hibshoosh H, Cheung K, Friedman RA, Yang CS, Milne GL, Liu DD, Lee JJ, Abdul K, Bigg M, Foreman J, Su T, Wang X, Ahmed A, Neugut AI, Akpa E, Lippman SM, Perloff M, Brown PH, Lightdale CJ
(2015) Cancer Prev Res (Phila) 8: 1131-7
MeSH Terms: Aged, Aged, 80 and over, Barrett Esophagus, Biopsy, Catechin, Chromatography, High Pressure Liquid, Dose-Response Relationship, Drug, Double-Blind Method, Esophagoscopy, Female, Humans, Male, Middle Aged, Phytotherapy
Show Abstract · Added March 10, 2016
This study was conducted to determine the safety and efficacy of the green tea-derived Polyphenon E (Poly E) in patients with Barrett's Esophagus (BE). Subjects were randomized to a 6-month, twice daily (BID) oral treatment of placebo or Poly E (200, 400, or 600 mg). Endoscopic evaluation, including biopsies, was performed before and after treatment. The primary objective was to demonstrate safety; secondary objectives investigated catechin accumulation and effects in clinical specimens. Of the 44 enrolled subjects, 11 received placebo, and 33 received Poly E. No dose-limiting toxicities were encountered, and a maximum tolerated dose (MTD) was not reached. The recommended phase II dose was 600 mg twice daily. The most common treatment-related adverse events (AE) in Poly E-treated subjects were grade I and II nausea, grade I belching, and grade I lactate dehydrogenase (LDH) elevation. No treatment-related AEs were reported in placebo-treated subjects, aside from grade I laboratory abnormalities. Pill counts and subject diaries were not consistently collected, and compliance was difficult to determine. However, on the basis of an intention-to-treat analysis, there was a significant relationship between Poly E dose and esophageal EGCG level--mean changes (pmol/g) of 0.79 (placebo), 6.06 (200 mg), 35.67 (400 mg), and 34.95 (600 mg); P = 0.005. There was a possible relationship between Poly E dose and urine PGE-M concentration. In conclusion, Poly E was well-tolerated, and treatment with Poly E (400 and 600 mg) but not Poly E (200 mg) or placebo resulted in clinically relevant and detectable EGCG accumulation in the target organ, esophageal mucosa.
©2015 American Association for Cancer Research.
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14 MeSH Terms
Environmental Interactions and Epistasis Are Revealed in the Proteomic Responses to Complex Stimuli.
Samir P, Rahul , Slaughter JC, Link AJ
(2015) PLoS One 10: e0134099
MeSH Terms: Chromatography, High Pressure Liquid, Epistasis, Genetic, Genotype, Glucose, Glycerol, Mass Spectrometry, Proteome, RNA, Messenger, Saccharomyces cerevisiae
Show Abstract · Added February 15, 2016
Ultimately, the genotype of a cell and its interaction with the environment determine the cell's biochemical state. While the cell's response to a single stimulus has been studied extensively, a conceptual framework to model the effect of multiple environmental stimuli applied concurrently is not as well developed. In this study, we developed the concepts of environmental interactions and epistasis to explain the responses of the S. cerevisiae proteome to simultaneous environmental stimuli. We hypothesize that, as an abstraction, environmental stimuli can be treated as analogous to genetic elements. This would allow modeling of the effects of multiple stimuli using the concepts and tools developed for studying gene interactions. Mirroring gene interactions, our results show that environmental interactions play a critical role in determining the state of the proteome. We show that individual and complex environmental stimuli behave similarly to genetic elements in regulating the cellular responses to stimuli, including the phenomena of dominance and suppression. Interestingly, we observed that the effect of a stimulus on a protein is dominant over other stimuli if the response to the stimulus involves the protein. Using publicly available transcriptomic data, we find that environmental interactions and epistasis regulate transcriptomic responses as well.
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9 MeSH Terms
Dietary isoflavones, urinary isoflavonoids, and risk of ischemic stroke in women.
Yu D, Shu XO, Li H, Yang G, Cai Q, Xiang YB, Ji BT, Franke AA, Gao YT, Zheng W, Zhang X
(2015) Am J Clin Nutr 102: 680-6
MeSH Terms: Adult, Aged, Asian Continental Ancestry Group, Case-Control Studies, Chromatography, High Pressure Liquid, Diet, Female, Follow-Up Studies, Humans, Isoflavones, Middle Aged, Multivariate Analysis, Nutrition Assessment, Prospective Studies, Risk Factors, Soybeans, Stroke, Surveys and Questionnaires
Show Abstract · Added May 4, 2017
BACKGROUND - Hormone therapy has been shown to increase risk of ischemic stroke in women. Plant-derived estrogens, particularly soy isoflavones, are known to have some estrogenic effects and have been marketed as natural alternatives to hormone therapy. Concerns have been raised about whether high isoflavone exposure may be related to ischemic stroke risk as well.
OBJECTIVE - We examined the dietary intake of isoflavones and the urinary excretion of isoflavonoids in relation to risk of ischemic stroke in women.
DESIGN - A prospective cohort study was conducted in 66,832 Chinese women (aged 40-70 y) who had no cardiovascular disease or cancer at baseline. Usual dietary intakes were assessed via in-person interviews with the use of a validated food-frequency questionnaire. Incident strokes were ascertained during follow-up home visits and confirmed by medical records. We also conducted a nested case-control study in postmenopausal women who had never used hormone therapy, including 1422 incident ischemic stroke cases and 1422 controls individually matched by age, date and time of urine sample collection, time since last meal, and use of antibiotics. Urinary isoflavonoids were measured with the use of high-performance liquid chromatography coupled with mass spectrometry.
RESULTS - During a mean follow-up of 10 y, 3110 incident ischemic strokes were verified. Dietary isoflavone intake was associated with increased risk of ischemic stroke; multivariable-adjusted HRs from lowest to highest quintiles were 1.00, 1.05, 1.10, 1.11, and 1.24, respectively (95% CI: 1.08, 1.42; P-trend = 0.002). In the case-control study, a similar positive association was observed for dietary isoflavones, but no significant associations were shown for the urinary isoflavonoid concentration [OR: 1.01 (95% CI: 0.77, 1.32) for comparison of extreme quintiles].
CONCLUSIONS - A habitually high intake of soy isoflavones may be associated with a modest but significant increase in risk of ischemic stroke in women. However, no association was shown for the urinary excretion of isoflavonoids.
© 2015 American Society for Nutrition.
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18 MeSH Terms
Polymerase Bypass of N(6)-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene.
Kotapati S, Wickramaratne S, Esades A, Boldry EJ, Quirk Dorr D, Pence MG, Guengerich FP, Tretyakova NY
(2015) Chem Res Toxicol 28: 1496-507
MeSH Terms: Butadienes, Chromatography, High Pressure Liquid, DNA, DNA Adducts, DNA Primers, DNA Replication, DNA-Directed DNA Polymerase, Deoxyadenosines, Epoxy Compounds, Humans, Kinetics, Oligodeoxyribonucleotides, Spectrometry, Mass, Electrospray Ionization
Show Abstract · Added March 14, 2018
N(6)-(2-Hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N(6)-HB-dA I) and N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N(6),N(6)-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N(6) position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N(6)-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N(6)-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N(6)-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N(6)-HB-dA I and (R,R)-N(6),N(6)-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N(6)-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N(6),N(6)-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N(6)-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N(6),N(6)-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N(6)-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N(6),N(6)-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N(6)-HB-dA I lesions are not miscoding, but that exocyclic (R,R)-N(6),N(6)-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.
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13 MeSH Terms
Analysis of protein composition using multidimensional chromatography and mass spectrometry.
Link AJ, Washburn MP
(2014) Curr Protoc Protein Sci 78: 23.1.1-23.1.25
MeSH Terms: Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Chromatography, Reverse-Phase, Mass Spectrometry, Proteins, Proteomics
Show Abstract · Added February 20, 2015
Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in-gel digestions by directly identifying protein mixtures in solution. One method used extensively is named Multidimensional Protein Identification Technology (MudPIT), where reversed-phase chromatography and strong cation-exchange chromatography are coupled directly in a microcapillary column. This column is then placed in line between an HPLC and a mass spectrometer for complex mixture analysis. MudPIT remains a powerful approach for analyzing complex mixtures like whole proteomes and protein complexes. MudPIT is used for quantitative proteomic analysis of complex mixtures to generate novel biological insights.
Copyright © 2014 John Wiley & Sons, Inc.
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6 MeSH Terms
The arginine decarboxylase pathways of host and pathogen interact to impact inflammatory pathways in the lung.
Paulson NB, Gilbertsen AJ, Dalluge JJ, Welchlin CW, Hughes J, Han W, Blackwell TS, Laguna TA, Williams BJ
(2014) PLoS One 9: e111441
MeSH Terms: Agmatine, Animals, Biofilms, Carboxy-Lyases, Chromatography, High Pressure Liquid, Disease Models, Animal, Female, Host-Pathogen Interactions, Humans, Inflammation, Longitudinal Studies, Lung, Mice, Mice, Inbred BALB C, Mutagenesis, Mutation, NF-kappa B, Nitric Oxide Synthase, Phenotype, Pneumonia, Bacterial, Prospective Studies, Pseudomonas aeruginosa, Sputum
Show Abstract · Added February 19, 2015
The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.
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23 MeSH Terms
Propofol detection and quantification in human blood: the promise of feedback controlled, closed-loop anesthesia.
Kivlehan F, Chaum E, Lindner E
(2015) Analyst 140: 98-106
MeSH Terms: Anesthesia, Chromatography, High Pressure Liquid, Electrochemical Techniques, Humans, Propofol
Show Abstract · Added June 11, 2018
The performance of a membrane-coated voltammetric sensor for propofol (2,6-diisopropylphenol) has been characterized in long term monitoring experiments using an automated flow analytical system (AFAS) and by analyzing human serum and whole blood samples by standard addition. It is shown that the signal of the membrane-coated electrochemical sensor for propofol is not influenced by the components of the pharmaceutical formulation of propofol (propofol injectable emulsion). The current values recorded with the electrochemical propofol sensor in buffer solutions and human serum samples spiked with propofol injectable emulsion showed excellent correlation with the peak heights recorded with an UV-Vis detector during the HPLC analysis of these samples (R(2) = 0.997 in PBS and R(2) = 0.975 in human serum). However, the determination of propofol using the electrochemical method is simpler, faster and has a better detection limit (0.08 ± 0.05 μM) than the HPLC method (0.4 ± 0.2 μM). As a first step towards feedback controlled closed-loop anesthesia, the membrane-coated electrochemical sensor has been implemented onto surface of an intravenous catheter. The response characteristics of the membrane-coated carbon fiber electrode on the catheter surface were very similar to those seen using a macroelectrode.
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