Propionate bioconversion to methane
Ontology highlight
ABSTRACT:
ORGANISM(S): Prokaryotic Environmental Samples
SUBMITTER: 
Yinguang Chen
PROVIDER: PXD032020 | iProX | Thu Mar 03 00:00:00 GMT 2022
REPOSITORIES: iProX
Similar Datasets
Project description:Samples were reduced by the addition of TCEP (25 mM final concentration) and incubated at 37 ºC for 10 min. Alkylation was carried out by the addition of iodoacetamide (25 mM final concentration) and incubated at RT for 1 h in the dark. Samples were then digested by the addition of 1:50 LysC (Wako, Japan) in 100 mM triethylammonium bicarbonate (TEAB) followed by incubation at 37 ºC for 6h and then addition of 1:50 trypsin (Thermo) followed by incubation at 37 ºC overnight. The efficiency of sample digestion efficiency was assessed by MS (LTQ XL, Thermo). The samples were then vacuum-dried and resuspended in 100 mM TEAB (100 µL). Samples were then incubated in their respective Tandem Mass Tag™ (TMT) 10-plex reagents (Thermo) for 1 h at RT with agitation. Reactions were quenched by the addition of 5% hydroxylamine for 15 min and each set of samples (treated and vehicle) were pooled in the same tube and dried overnight. The TMTTM-labelled samples were dried and kept at -85 ºC until further analysis.
2021-07-18 | PXD023780 | Pride
Project description:We applied a non targeted mass spectrometric assay (LCMSE), to quantify 55 abundant plasma proteins in specimens from 31 healthy volunteers. Quantitation was done by HI3 peptide measurement using a single internal standard to estimate protein concentrations. Results for 7 apolipoproteins were compared with those obtained using isotope labelled standards, while 12 proteins were compared to routine immunoassays. Blood samples were obtained from 31 healthy volunteers (17 males; 14 females) with a median age of 46 years and a range of 22-67 years. Total plasma protein concentration was assayed with a BCA-assay (20) according to the manufacturer’s protocol (Thermo). Samples were diluted tenfold in 0.1% Rapigest SF (Waters Corporation, Milford, MA), 50 mM ammonium bicarbonate and heated at 95° C for 15 min. Subsequently, plasma samples were reduced with 5 mM dithiothreitol (60° C, 30 min) and alkylated with 15 mM iodoacetamide (ambient temperature, dark, 30 min). Proteolytic digestion was performed with modified trypsin (gold grade, Promega, Madison WI) at 0.3 units/µg protein, (37° C, 20 hours) unless indicated otherwise. Following digestion, Rapigest SF was broken down by adding 1% trifluoroacetic acid (pH<2, 37 °C, 45 min). Peptide solutions were centrifuged (20,000 x g, 10 min) and supernatant was collected. Prior to analyses a MASSPREP protein digestion standard (Waters Corporation, ADH1 or ENO from Saccharomyces cerevisiae) was added for quantitation purposes. LC-MS analyses were performed using ~ 0.21 µg of the final plasma protein digest mixtures (384 times total dilution) unless indicated otherwise. LC separations of tryptic peptides were performed with a NanoAcquity system (Waters Corporation), coupled to a Synapt G2 quadrupole time of flight mass spectrometer (Waters Corporation, Manchester, UK). Accurate mass precursor and fragment ion LC-MS data were collected in data independent LCMSE mode of acquisition. Continuum LC-MS data were processed using ProteinLynx GlobalSERVER version 2.5 (PLGS 2.5, Waters Corporation). Parameter settings: digest reagent trypsin, allow 1 ‘missed cleavage’, search tolerances automatic, typically 5 ppm for precursor and 15 ppm for product ions, fixed modification cysteine carbamidomethylation, and variable modification methionine oxidation. Protein identifications were obtained searching the human SwissProt entries of an UniProt database (release 13.2). This database was modified to include N-terminal processing of proteins using protein maturation device software, with ADH1 and ENO1 of S. cerevisiae appended as internal standard to address technical variation and allow concentration determinations. Estimation of false-positive identification rates was done by searching a randomized version of the abovementioned human protein database generated within PLGS 2.5. Data were exported as csv-files for further, detailed analysis. Stringent criteria were applied for quantitation, protein identifications were only considered significant if reported in 14 or more samples. Protein false positive identification rates were estimated using the criteria mentioned above and no false positives were identified in these searches. DATASET: KC1-31: 31 healthy volunteers QC1-10: 10 times injection of a single sample to ascertain analytical variation over 9 days of measurements. 20120802_QC1-QC6: 6 pooled plasma samples worked up and analysed during a single day of measurements to ascertain Intra-assay variation. 20120427_S2,S4;20120525_S18,S19;20120626_S3,S4;20120802_QC7: 7 pooled plasma samples processed and analysed during 3 months of routine measurements to analyse Inter-assay variation 20130416_s1-s5: pooled plasma samples spiked with a QCONCATAMER for 11 apolipoproteins labelled by heavy Lysine and Arginine (+6.02 AMU) to quantify apolipoproteins by internal isotope labelled standard.
2015-10-20 | PXD000347 | Pride
Project description:Using cell sorting technique, we collected Arabidopsis root hair protoplasts (transgenic line harboring root hair specific gene promoter ::GFP, here named GFP) and nonGFP protoplasts( whole root except root hair). Then, we extracted total proteins and RNA for proteome and RNASeq. RNAseq data have already been deposited into NCBI (accession numbers SRR364677 and SRR364678). Total proteins were separated by 1D SDS-PAGE, followed by gel slicing, in gel digested and run ESI-LC-MS/MS on LTQ Orbitrap Velos. We set two biological repeats and each sample was scanned two times (two technique repeats). We then combined all ms/ms raw data from all fractions and technique repeats from one biological sample into one file to indentify proteins by searching Arabidopsis protein database with software Proteome Discoverer (with two searching engines mascot and sequester). Protocol: Protoplasts from EXP7 and non-GFP protoplastswere stored at -80°C, thawed on ice for 5 min, vortexed several times and suspended in 5 x volume of pre-cooled acetone (-20°C) containing 10% (v/v) trichloroacetic acid (TCA) and 0.07% (v/v) 2-mercaptoethanol. Proteins were then precipitated for 2 h at −20°C after thorough mixing. Proteins were collected by centrifuging at 35,000 g (JA-20 108 rotor, Beckman J2-HS) at 4°C for 30 min. The supernatant was removed and the protein pellets were washed three times with cold acetone containing 0.07% (v/v) 2-mercaptoethanol and 1 mMphenylmethanesulfonylfluoride. Protein pellets were dried by lyophilization and stored at -80°C, or immediately extracted using Laemmli buffer (63 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue). Protein concentration was determined using Pierce protein assay kit (Pierce, Rockford). Proteins from each sample were separated using a Bis-Tris precast gradient gel (4−16%, Bio-Rad) according to the manufacturer’s instructions. After electrophoresis, the gel was Coomassie-stained using the Colloidal Blue Staining Kit (Invitrogen). The protocol used for in-gel trypsin digestion of proteins in gels was adapted from the method described in Shevchenko et al. (1996). Briefly, protein bands were manually excised and each band was cut into small pieces (~0.5 mm). The gel pieces were washed three times with a solution containing 50% methanol and 5% acetic acid for 2 h, twice with a solution containing 25 mM NH4HCO3 in 50% acetonitrile for 10 min each, and then the gel pieces were dried in a vacuum centrifuge. Reduction of proteins in gel pieces was performed with DTT and alkylation with iodoacetamide, and the gel pieces were washed and dried in a vacuum centrifuge. A trypsin solution in 25 mM NH4HCO3 containing 75 to 100 ng of sequencing grade modified trypsin (Promega) in 25-40 µl was added and incubated with gel pieces for 12-16 h at 37°C. To recover the tryptic peptides, a solution of 30 µl containing 5% formic acid and 50% acetonitril was added to the gel pieces, agitated in a vortex for 30-60 min and withdrawn into a new tube. The recovery was repeated once with 15 µl solution and the resulting two recovers were combined and dried in a vacuum centrifuge. The dried pellet was re-dissolved in 10-20 µl of 0.1% formic acid for LC-MS/MS analysis.
2013-05-28 | PXD000195 | Pride
Project description:For gaining additional insights into the composition of the testicular proteome of the domestic pig (Sus scrofa domestica), we conducted 2DE-MS. Two-dimensional SDS PAGE was run on testicular lysates of three boars, with three gels per boar. Upon matching across gels, we arbitrarily selected protein spots for mass spectrometry analysis. Excised slices were vacuum dried and soaked with digestion buffer containing trypsin (0.01 μg/μl), followed by overnight incubation at 37°C in the same buffer without trypsin. Subsequently, peptides were extracted in solvents of increasing acetonitrile content, by sonication. Upon vacuum-centrifugation, peptides were reconstituted in 0.1% formic acid (FA). Following this, peptides were fractionated by reversed phase liquid chromatography (C18; buffer A: 0.1% FA dissolved in HPLC-H2O; buffer B: 0.1% FA, dissolved in CAN; flow-rate: 0.4 µL/min; gradient: 2-30% in 30 minutes). Eluted peptides were injected via an electrospray ionization interface into a Q-TOF mass spectrometer (one boar, Q TOF Ultima, Micromass/Waters, Manchester, UK) and an ion-trap mass spectrometer (two other boars, XCT ion-trap, Agilent Technologies, Waldbronn, Germany). We used ProteomeDiscoverer 2.4 (Thermo Fisher Scientific, San Jose, USA) for peptide and protein identification. Using Sequest HT, we searched peak lists (*.mgf) against the Sus scrofa reference proteome database (UniProt Proteome ID: UP000008227, 49,793 proteins).
2022-08-12 | PXD015649 | Pride
Project description:Cervicovaginal lavage (CVL)supernatants were heat inactivated for 30 minutes at 56°C before further processing. Total protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific, Rockford, IL, USA). Sample protein content and volume were normalised with 25mM ammonium bicarbonate (ABC). Soluble proteins were precipitated using an equal volume of ice cold 30% (w/v) TCA in acetone and incubated at -20⁰C for 2 hours. Samples were centrifuged at 12,000g for 10 minutes (4⁰C) to pellet proteins. Pellets were washed three times with ice cold acetone and allowed to air dry. Further sample processing was performed as previously described (Armstrong et al, 2014) with minor modifications. Briefly, protein pellets were resuspended in 25 mM ABC, 0.05%(w/v) rapigest (Waters), reduced and alkylated. Digestion was performed with proteomic-grade trypsin (Sigma-Aldrich, St. Louis, MO, USA) at a protein:trypsin ratio of 50:1. Rapigest was precipitated by addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the nanoACQUITY-nLC system (Waters MS technologies) coupled to an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) mass spectrometer equipped with the manufacturer’s nanospray ion source. The gradient of the analytic column (nanoACQUITY UPLCTM BEH130 C18 15cm x 75µm, 1.7µm capillary column) consisted of 3-40% acetonitrile in 0.1% formic acid for 90 min then a ramp of 40-85% acetonitrile in 0.1% formic acid for 5min.
2016-03-15 | PXD003176 | Pride
Project description:Exponentially growing S.aureus ATCC 25923 was treated with linezolid (LZD) at concentrations of 1/2× MIC, 1× MIC, and 5× MIC, or an equivalent amount of DMSO for 1 hour. The bacteria were pelleted by centrifugation and washed twice with saline. The samples were then re-suspended in bacteria lysis buffer (100 mM NH4HCO3, 8 M urea, 1 mM PMSF, 1× Protease Inhibitor Cocktail) and mechanically lysed using 0.1 mm silica beads in a Precellys tissue homogenizer at 4°C. Subsequently, the samples were crushed with a non-contact ultrasonic cell crusher for 1 hour. Silica beads and bacterial debris were removed by centrifugation at 1200×g and 4°C for 20 minutes. The protein concentration in the supernatant was measured using a Bradford assay kit (Beyotime, China). Next, the samples were reduced with 10 mM DTT for 1 hour, followed by alkylation in the dark with 45 mM iodoacetamide to break disulfide bonds. Each sample underwent trypsin digestion, with 50 μg of total protein digested using 2 μg trypsin overnight at 37°C or 1.5 hours at 56°C, and an additional 1 μg trypsin added twice during digestion. The digested peptides were filtered using Ultra Centrifugal Filters (Millipore, USA) and desalted using MonoSpin C18 (Shimadzu, Japan) following the manufacturer's protocol. The peptide samples were dried using a freeze-dryer and resuspended in 25 μl of water with 0.1% formic acid (vol/vol).Data acquisition was performed using a reverse-phase LC-MS/MS system comprising an Easy nLC1200 system (ThermoFisher, USA) coupled with a Q Exactive Plus Orbitrap MS System (ThermoFisher, USA).
2023-12-11 | PXD046938 | Pride
Project description:Briefly, each cecum was ground to powder in liquid nitrogen, dissolved in lysis buffer with 150 µL of radioimmunoprecipitation assay buffer containing 1 mM PMSF and a protease inhibitor cocktail (CST, USA), and then subjected to sonicating. The cellular debris was removed by centrifugation. The concentration of the extracted protein was determined and the quality was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The extracted protein was digested into peptides using trypsin Gold at a protein: trypsin ratio of 30:1 at 37oC for 16h. Following trypsin digestion, the peptides were desalted on a Strata X C18 SPE column, vacuum-dried, resuspended in 0.5M TEAB. Each peptide mixture was labeled using the 8-plex iTRAQ reagent kit (Applied Biosystems, Foster City, USA). The labeled samples were mixed, dried, resuspended and then loaded onto a MacroSpin VydacC18 reverse-phase minicolumn . Following washing and elution, the samples were dried and fractionated using a strong cation exchange column. Each SCX fraction was lyophilized and dissolved in the solvent and 0.01% trifluoroacetic acid. The peptides were loaded onto a C18 capillary trap cartridge and then separated on a 15-cm nanoflow C18 column at a flow rate of 200 nL/min using a Proxeon EASY-nLC system. The peptides were eluted from the HPLC column using a linear gradient including 5% solvent B for 10 min, 5-30% solvent B for 30 min, 30-60% solvent B for 5 min, 60-80% solvent B for 5 min and holding for 5 min, and 80-5% solvent B for 5 min, after which they were maintained in 5% solvent B for 5 min. The flow from the column was directed to a tandem mass spectrometer in the TripleTOF 5600 system operating in positive ion mode. Tandem mass spectra were searched against the chicken protein database (http://www.uniprot.org/proteomes/UP000000539) using ProteinPilotTM V4.5 for computational analysis.
2023-12-01 | PXD047433 |
Project description:In this work, we investigated changes in protein structures in vacuum-packed pork during chill storage and its impact on in vitro protein digestion. Longissimus dorsi muscles were vacuum packed and stored at 4°C for 3 days. Samples were subjected to Raman spectroscopy, in vitro digestion and Nano LC-LTQ-Orbitrap XL MS/MS. The 3 d samples had lower α-helix content, but higher β-sheet, β-turn and random coil contents than the 0 d samples (P < 0.05). SDS-PAGE revealed significant protein degradation in the 3 d samples and the differences in digested products across storage time. Proteome analysis indicated that the 3 d samples had the higher susceptibility to digestion. Increasing protein digestibility was mainly attributed to the degradation of myofibrillar proteins. Thus, exposure of more enzymatic sites in loose protein structure during chill storage could increase protein degradation in meat.
2021-09-08 | PXD020595 | Pride
Project description:LC-MS/MS analysis of peptide digests of myofibrils from wild type and Dmlc2(Delta 2-46; S66A, S67A) mutant Drosophila flies. Myofibrils and thick filament samples were solubilized in 150 ul 0.1% RapiGest SF Surfactant (Waters Corporation; 50 C, 1 hour). Proteins were reduced by addition of 0.75 ul of 1M dithiothreitol and heating (100 C, 10 min). Cysteines were alkylated by addition of 22.5 ul of 100 mM iodoacetamide in 50 mM ammonium bicarbonate and incubation in the dark (22C, 30 min). Proteins were digested to peptides by addition of 25 ul of 50 mM ammonium bicarbonate containing 5 ug of trypsin (Promega) and incubating (37 C, 18 hours). The samples were dried down in speed vacuum device. Trypsin was deactivated and RapiGest was cleaved by addition of 100 ul of 7% formic acid in 50 mM ammonium bicarbonate and heating (37 C, 1 hour). The resultant peptides were dried down. RapiGest was cleaved again by addition of 100 ul of 0.1% trifluoroacetic acid and heating (37 C, 1 hour). The resultant peptides were dried down and reconstituted a final time in 60 ul of 0.1% trifluoroacetic acid. The samples were centrifuged at 18,800 RCF for 5 minutes (Thermo, Sorvall Legend Micro 21R) to pellet the surfactant. The top 57 ul of solution was transferred into a mass spectrometry (MS) analysis vial.
A 20 ul aliquot of each sample was injected onto an Acquity UPLC HSS T3 column (100 A, 1.8 um, 1 x 150 mm) (Waters Corporation) attached to a UltiMate 3000 ultra-high pressure liquid chromatography (UHPLC) system (Dionex). The peptides were separated and the UHPLC effluent was directly infused into a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (MS) through an electrospray ionization source (Thermo Fisher Scientific). Data were collected in data-dependent MS/MS mode with the top five most abundant ions being selected for fragmentation.
2020-06-24 | MSV000085627 | MassIVE
Project description:Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.
2023-07-20 | PXD040867 | Pride