Dataset Information

Complete Expressed Proteome of Bartonella Henselae

ABSTRACT: Using a discovery proteomics approach, the expressed proteome of Bartonella henselae, a Gram-negative prokaryotic model organism was exhaustively studied under two conditions that mimic those encountered in different hosts. Using the analysis-driven experimentation (ADE) feedback-loop strategy, we were able to virtually eliminate the biases of commonly under-represented short, basic, and particularly lower abundant and membrane protein classes, all of which are experimentally tractable. Based on a very stringent FDR at the PSM level, we identified 85% of all distinct, annotated proteins and above 90% compared to the expressed protein-coding genes in the two conditions. Several lines of evidence indicated that this is very close to all proteins that can be identified by a discovery proteomics approach with current technology Our experimental strategy relied on four elements: First, we used a combination of sub-cellular fractionation (Cyt, TM, IM, OM) with additional biochemical fractionation regimens (gelfiltration, OGEpep, OGEprot, ProteoMiner) to reduce the overall sample complexity. Second, an exclusion list approach was applied, where each sample is measured twice in a mass spectrometer. Third, we relied on the ADE strategy to target typically under-represented areas of the proteome. Finally, we also used chymotrypsin as enzyme in addition to trypsin for all membrane-derived fractions. For computational analysis, we combined results from two database search engines (Mascot and MS-GF+).Mass spectra were searched against a protein sequence database containing 1488 NCBI RefSeq annotated B. henselae proteins (NC_005956.1), 3336 sheep proteins, a positive control (myc-gfp), as well as protein sequences of 256 common contaminants. Spectra were searched against this database using the target/decoy option either with Mascot (version 2.3.0, Matrix Science) or with MS-GF+ (MS-GFDB v7747, kindly provided by Dr. Sangtae Kim, UCSD, USA) using the following parameters: Carbamidomethylation was set as a fixed modification on all Cysteines, oxidation of Methionines, deamidation of Asparagines and Glutamines, as well as cyclization of N-terminal Glutamines were considered as optional modifications. Spectra were searched for a match to fully-tryptic and semi-tryptic peptides with up to two missed cleavage sites. Precursor ion mass tolerance was set to 5 ppm, fragment ion mass tolerance was set to 0.8 Da, and the automatic decoy search option was enabled. For Mascot, data were further post-processed with Percolator (Brosch et al. 2009). Additional RNA-seq data are at GEO under the accession: GSE44564.

OTHER RELATED OMICS DATASETS IN: PRJNA190078

INSTRUMENT(S): LTQ FT, LTQ Orbitrap

ORGANISM(S): Bartonella Henselae (strain Atcc 49882 / Houston 1) (rochalimaea Henselae)

SUBMITTER: Ulrich Goldmann

PROVIDER: PXD000153 | Pride | 2013-07-18

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20011221_02_BarH_IM2_6to7.RAW	Raw
	20011221_03_BarH_IM2_8to9.RAW	Raw
	20011221_04_BarH_IM2_10to11.RAW	Raw
	20011221_05_BarH_IM2_12to13.RAW	Raw
	20011221_06_BarH_IM2_14to15.RAW	Raw

Items per page:

1 - 5 of 747

Publications

Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome.

Omasits Ulrich U Quebatte Maxime M Stekhoven Daniel J DJ Fortes Claudia C Roschitzki Bernd B Robinson Mark D MD Dehio Christoph C Ahrens Christian H CH

Genome research 20130722 11

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interacti ...[more]

PMID: 23878158

Similar Datasets

Project description:B. japonicum cells were grown either in minimal medium (BVM) with L-arabinose as carbon and energy source or in complete medium (PSY) containing L-arabinose. Proteins extracted from three replicates of B. japonicum cells grown in complex and minimal medium with L-arabinose until mid-exponential phase were separated on a Tris-HCl polyacrylamide gel. After reduction and carbamidomethylation, the proteins were digested with trypsin (Promega, Madison, WI, USA), and the resulting peptides were separated by RP-HPLC and analyzed by a hybrid LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) interfaced with a nanoelectrospray source. Mass spectra were further processed with an in-house processing pipeline. A total of 3,390 and 3,149 proteins were identified in complex and minimal medium respectively. We identified highly expressed products of candidate genes involved in the degradation of arabinose and suggest that glycolaldehyde is oxidized to glyoxylate and then reduced to glycerate through a glyoxylate carboligase and a tartronate semialdehyde reductase for assimilation and/or converted to oxalate and then oxidized to formate and CO2 through a formyl-CoA transferase (Frc) and an oxalyl-CoA decarboxylase (Oxc) for energy generation. Fragment ion mass spectra were extracted from Thermo RAW files using msconvert (ProteoWizard, version 3.0.3831), merged in one MGF file per gel and searched against a composite B. japonicum USDA 110 protein database (RefSeq NC_004463.1, July 22, 2013) containing 256 common contaminants (e.g. human keratin, trypsin) with the search engine MS-GF+ (MS-GFDB v7747) for a match to fully-tryptic and semi-tryptic peptides with up to two missed cleavage sites with a mass tolerance of 25ppm and with oxidation (M), deamidation (NQ), methylation (DE) as variable modifications and carbamidomethylation (C) as fixed modification. Using the decoy option of MS-GF+, we filtered the list of peptide spectrum matches (PSMs) to an estimated overall FDR of 0.2%, classified the PSMs with PeptideClassifier, and only further considered peptides (tryptic or semi-tryptic) that unambiguously imply one bacterial protein sequence. The FDR at the peptide level amounted to about 1.5%, that at the protein level to about 2.5% when requiring two peptides or three spectra for a protein identification.

Project description:Tistlia consotensis is a halotolerant Rhodospirillaceae that was isolated from a saline spring located in the Colombian Andes with a salt concentration close to seawater (4.5%w/vol). We cultivated this microorganism in three NaCl concentrations, i.e. optimal (0.5%), without (0.0%) and high (4.0%) salt concentration, and analyzed its cellular proteome. For assigning tandem mass spectrometry data, we first sequenced its genome and constructed a six reading frame ORF database from the draft sequence. We annotated only the genes whose products (872) were detected. We compared the quantitative proteome data sets recorded for the three different growth conditions.Peak lists were generated with the MASCOT DAEMON software (version 2.3.2) from Matrix Science using the extract_msn.exe data import filter from the Xcalibur FT package (version 2.0.7) proposed by ThermoFisher. Data import filter options were set at 400 (minimum mass), 5,000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1,000 (threshold). MS/MS spectra were searched against the home-made ORF database with the following parameters: tryptic peptides with a maximum of 2 miss cleavages during proteolytic digestion, a mass tolerance of 5 ppm on the parent ion and 0.5 Da on the MS/MS, fixed modification for carbamidomethylated Cys (+57.0215) and variable modification for oxidized Met (+15.9949). All peptide matches with a peptide score above its query threshold set at p < 0.05 with the ORF database and rank 1 were parsed using the IRMa 1.28.0 software. False-positive rate for peptide identification was estimated using a decoy database as below 0.5% with these parameters. MS/MS spectra assigned to several loci were systematically removed. A protein was considered validated when at least two different peptides were detected in the same experiment. False-positive identification of proteins was estimated using a reverse decoy database as below 0.1% with these parameters The number of MS/MS spectra per protein (spectral counts) was determined for the three replicates in each growth condition. The protein abundances were compared using the T-Fold option of the PatternLab 2.0 software. This module allows normalising the spectral count datasets, calculating the average fold changes with statistics (t-test), and estimating the resulting theoretical false discovery rate. Stringent parameters were used in this analysis: minimum fold change of 1.5, minimum p-value of 0.05 and BH-FDR Alfa of 0.15.

Project description:The use of internal calibrants (the so called lock mass approach) provides much greater accuracy in mass spectrometry based proteomics. However, the polydimethylcyclosiloxane (PCM) peaks commonly used for this purpose are quite unreliable, leading to missing calibrant peaks in spectra and correspondingly lower mass measurement accuracy. Therefore, we here introduce a universally applicable and robust internal calibrant, the tripeptide Asn3. We show that Asn3 is a substantial improvement over PCM both in terms of consistent detection and resulting mass measurement accuracy. Asn3 is also very easy to adopt in the lab, as it requires only minor adjustments to the analytical setup. Data analysis: For mass measurement accuracy (MMA) calculations and comparisons, the following Mascot workflow was used. From the MS/MS data in each LC run, Mascot Generic Files were created using Distiller software (version 2.4.3.3, Matrix Science, London, UK, www.matrixscience.com/distiller.html). These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.4.0, Matrix Science). Spectra were searched against the Swiss-Prot database (version 13_04 of UniProtKB/Swiss-Prot protein database containing 20,232 sequence entries of human proteins) concatenated with its reversed sequence database. Variable modifications were set to pyro-glutamate formation of amino terminal glutamine and acetylation of the protein N-terminus, whereas fixed modifications only included oxidation of methionine. Mass tolerance on peptide ions was set to 10 ppm (with Mascot’s C13 option set to 1), and the mass tolerance on peptide fragment ions was set to 20 millimass units (mmu), except for the space-charge effect experiment(LMA5) where an extra search was done with a setting of 3 mmu. The peptide charge was set to 1+,2+,3+ and instrument setting was put on ESI-QUAD. Enzyme was set to trypsin allowing for one missed cleavage, and cleavage was allowed when arginine or lysine is followed by proline. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. All data was processed and managed by ms_lims.