Dataset Information

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MS)

ABSTRACT: Absolute protein quantification for cytosolic proteins of B. subtilis growing on a carbon source (condition S) or on an amino acid source (condition CH). Data for 3 biological replicates and 3 technical replicates each for each condition are provided. For data processing and protein identification, raw data were imported into ProteinLynx Global Server 2.4(PLGS, Waters) and processed via Apex3D algorithm with following parameters:chromatographic peak width:automatic, MS ToF resolution: automatic, lock mass for charge 2:785.8426Da/e, lock mass window: 0.25Da, low energy threshold: 250cts, elevated energy threshold: 30cts, retention time window: automatic, intensity threshold of precursor/fragment ion cluster:1000cts. Database search was carried out by the ion accounting algorithm against randomized _B. subtilis_ 168 database with added common laboratory contaminants and yeast ADH1 sequence (8,438 entries) using following parameters: peptide tolerance:automatic, protein tolerance: automatic, min fragment ions matches per peptide: 1, min fragment ions matches per protein: 5, min peptide matches per protein: 2, primary digest reagent: trypsin, missed cleavages: 2, variable modifications: carbamidomethylation C (+57.0215), deamidation N, Q (+0.9840),oxidation M (+15.9949), pyrrolidonecarboxylacid N-TERM (-27.9949), false discovery rate (FDR): 5%, calibration protein: yeast ADH1. With PLGS absolute protein quantification is based on comparison of the summed signal intensities of the three most intense peptides of a protein to these of ADH1 (Hi3 approach).

INSTRUMENT(S): Synapt MS

ORGANISM(S): Bacillus Subtilis Subsp. Subtilis Str. 168

SUBMITTER: Sandra Maass

PROVIDER: PXD000154 | Pride | 2014-08-04

REPOSITORIES: Pride

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Publications

Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)).

Muntel Jan J Fromion Vincent V Goelzer Anne A Maaβ Sandra S Mäder Ulrike U Büttner Knut K Hecker Michael M Becher Dörte D

Molecular & cellular proteomics : MCP 20140131 4

In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MS(E)). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% cou ...[more]

PMID: 24696501

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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.

Project description:Total lysates were prepared from IRF3 KO 293T cells transfected with Flag-IRF3 and followed by immunoprecipitation with α-FLAG M2 beads. Immunoprecipitated proteins were separated by SDS-PAGE, and then stained with Coomassie Blue. The entire lane was excised, digested with trypsin, and analyzed with LC-MS/MS. LC-MS/MS identification of peptide mixtures was performed once with two replicates per condition at BIOPROFILE TECHNOLOGY (Shanghai, China). Briefly, peptides were chromatographed through Easy-nLC1200 (Thermo Fisher, California, USA). Peptide samples were loaded by Trap column (reverse-phase), (100 μm*2 cm, 5μm, C18, Dr. Maisch GmbH) and separated by Thermo scientific EASY column (75 μm*15cm, 3 μm, C18) at 300 nL min−1 for 60 min using a four-step acetonitrile (0.1% formic acid in 80% acetonitrile) gradient: 2–5% over the first 2 min and 5–28% for 2–44 min and 28–40% for 44–51 min and 40–100% for 51–53 min. The tandem mass spectrometry was performed by Q Exactive mass spectrometer (Thermo Fisher, California, USA). The MS1 survey scan (350–1800 m/z) was at a resolution of 60,000 at 200 m/z with automatic gain control (AGC) target of 3e6 and a maximum injection time of 50 ms. Dynamic exclusion was 60.0 s. Each full scan takes 20 MS2 scans. MS2 activation type was HCD model. Isolation window was 2 m/z. The MS2 survey was at a resolution of 15,000 at 200 m/z with automatic gain control (AGC) target of 1e5 and a maximum injection time of 50 ms. RAW files generated by spectrometer was subjected to Proteome Discoverer (version 1.4) software with searching the library of Uniprot homo sapiens (uniprot-Homo sapiens (Human) [9606]-204995-20220606.fasta) for protein identification. Trypsin was specified as the proteolytic enzyme, with up to two missed cleavage sites allowed. Carbamidomethylation was set as the fixed modification. Oxidation (M) and ubiquitination [GlyGly (K)] were set as the variable modifications. The precursor mass tolerance was set to 20 ppm and the fragment ion tolerance at 0.1 Da. Proteins were identified based on at least one unique peptide. Protein and peptide identification confidence threshold were set to an FDR of 1%. The tandem mass spectra of matched peptides were checked manually for their validity.

Dataset Information

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MS)

Publications

Comprehensive absolute quantification of the cytosolic proteome of Bacillus subtilis by data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)).

Similar Datasets

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