Dataset Information

Proteins from Outer Membrane Vesicules of Pseudomonas syringae Lz4W

ABSTRACT: The proteins from Outer Membrane Vesicles (OMVs) were extracted by chilled acetone method. The proteins were separated on 1D SDS-PAGE (12%). From the gel, total 10 fractions were made and all of them were subjected to in gel digestion using trypsin. The MS/MS spectra of the resulting tryptic peptides were recordedby using LC coupled ESI-MS/MS (Thermo Orbitrap Velos). The mass spectral data thus obtained was analysed by using Proteome Discoverer 1.3. Since the genome sequence of P. syringae Lz4W is not available, the data was searched against a database prepared from 20 related Pseudomonas species whose genome sequence is available on Uniprot (Updated upto Jan 2013). The search was observed by using nodes Sequest and Mascot both, the enzyme selected was trypsin with maximum 2 missed cleavages, the precursor tolerance set at 10 ppm, fragment tolerance at 0.8 Da, carbamidomethylated cystein (57.02 Da) as fixed modification, oxidised methionine (15.99 Da) as variable modification. After the search is over, the results were refined applying result filters as Peptide confidence (High) and Differentiable Proteins (including distinct proteins), which makes sure that each protein entry in the list is identified with at least one unique peptide.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Pseudomonas Sp.

SUBMITTER: Medicharla V. Jagannadham

PROVIDER: PXD000221 | Pride | 2014-01-29

REPOSITORIES: Pride

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Publications

Molecular characterization and functional analysis of outer membrane vesicles from the antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions.

Kulkarni Heramb M HM Swamy Ch V B ChV Jagannadham Medicharla V MV

Journal of proteome research 20140129 3

Outer membrane vesicles (OMVs) of Gram-negative bacteria form an important aspect of bacterial physiology as they are involved in various functions essential for their survival. The OMVs of the Antarctic bacterium Pseudomonas syringae Lz4W were isolated, and the proteins and lipids they contain were identified. The matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) analysis revealed that phosphatidylethanolamines and phosphatidylglycerols are the main lipid components. Th ...[more]

PMID: 24437924

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Project description:Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using Acyl-ATP (AcATP) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Protein extracts were labelled with NHS-LC-Biotin, purified over neutravidin and eluted with formic acid. Eluted proteins were separated by 1-D SDS Gel and digested with Trypsin. Peptide mixtures were analysed by LC-MS/MS on an LTQ Velos. Spectra were searched using Mascot 2.3 using a fixed modification of cysteine (57.02 Da for carbamidomethyl), and variable modifications of methionine (15.99 Da for oxidation) and lysine (339.16 and 355.16 Da for BHAc and OBHAc labeling, respectively). A mass tolerance was set to 0.3 Da for the precursor ions and 0.4 Da for fragment ions. Up to two mis-cleavages for trypsin were permitted since the labeling would prevent cleavage at the labeled lysine. The MS2 spectra were searched against the TAIR10_pep_2012 (version November 2012) containing 35386 proteins of Arabidopsis thaliana, supplemented with a small database with 1095 artefact proteins and a reversed decoy database of the same proteins (totals 72962 entries). An oxidized version of the modifier was observed. Alternatively Arabidopsis leaf proteome was labeled with desthiobiotin-acyl ATP and digested with trypsin. Labeled peptides were purified on streptavidin matrix and sequenced by LC-MS/MS on an LTQ linear ion trap. Here the MS2 spectra were searched using SEQUEST against the TAIR9_pep_20090619 database (version June 2009) containing 32,769 proteins of Arabidopsis thaliana. MS2 searches included fixed iodoacetamide modification of cysteine (57 Da for alkylation), and variable modifications of methionine (16 Da for oxidation) and lysine (196 Da for DBAcATP labeling). Up to three mis-cleavages with trypsin were allowed and non-tryptic or half-tryptic peptides were excluded. A mass tolerance was set to 3 Da for the precursor ions and 1 Da for fragment ions.

Dataset Information

Proteins from Outer Membrane Vesicules of Pseudomonas syringae Lz4W

Publications

Molecular characterization and functional analysis of outer membrane vesicles from the antarctic bacterium Pseudomonas syringae suggest a possible response to environmental conditions.

Similar Datasets

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