Dataset Information

Determination of Phosphorylation Sites in the DivIVA Protein of Streptomyces coelicolor by Targeted LC-MS/MS

ABSTRACT: The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, additional tryptic cleavage sites were introduced into DivIVA and the resulting phosphopeptides were analysed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC-MS/MS and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers, and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on 5 sites in the C-terminal part of the protein (T304, S309, S338, S344 and S355).

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Streptomyces Coelicolor

SUBMITTER: Gerhard Saalbach

PROVIDER: PXD000095 | Pride | 2013-08-05

REPOSITORIES: Pride

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Publications

Determination of phosphorylation sites in the DivIVA cytoskeletal protein of Streptomyces coelicolor by targeted LC-MS/MS.

Saalbach Gerhard G Hempel Antje M AM Vigouroux Marielle M Flärdh Klas K Buttner Mark J MJ Naldrett Michael J MJ

Journal of proteome research 20130815 9

The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of ...[more]

PMID: 23905541

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Project description:The proteolytic activation of protein kinase C delta generates a catalytic fragment called PKC delta-CF, which induces cell death. However, the mechanisms underlying PKC delta-CF-mediated cell death are largely unknown. On the basis of an engineering leukemic cell line with inducible expression of PKC delta-CF, here we employ SILAC-based quantitative phosphoproteomics to systematically and dynamically investigate the overall phosphorylation events during cell death triggered by PKC delta-CF expression. Totally, 3000 phosphorylation sites were analyzed. We combined SILAC for quantitation, strong-cation exchange chromatography and titanium dioxide chromatography for phosphopeptide enrichment, and high-accuracy mulistage MS. All MS/MS ion spectra were analyzed using Sequest version v.27, rev. 1146 (Thermo Fisher Scientific, San Jose, CA) which were incorporated into the Sorcerer engine version 4.0.4 build (Sage-N Research). Sequest were set up to search the ipi.HUMAN.v3.65 database (86379 entries) (ftp.ebi.ac.uk/pub/data bases/IPI/current) with its target-decoy database as a reversed complement assuming the semi-enzyme tryptic digestion allowed for three missed tryptic cleavages with full mass from 600 to 4600. The precursor ion tolerance was set to ±10 p.p.m and MS2 ions tolerance was set to 1 Da. Searches parameters for non-phosphopeptides were allowed for a static modification of Carbamidomethyl cysteine (C, +57.021465) and dynamic modifications of oxidized methionine (M, +15.99492) on, SILAC-labeled lysine (K, 6C2N, +8.014199) on and SILAC-labeled arginine (R, 6C4N, +10.008269) with up to 4 total dynamic modifications and up to 3 of any particular dynamic modification. In addition, searches for phosphopeptides were allowed for dynamic modifications of phosphorylation serine (S), threonine (T), and tyrosine (Y) (+79.966331) with up to 6 total dynamic modifications and up to 4 of any particular dynamic modification. The ASCORE algorithm was selected to assess the possibility of phosphorylation sites. DATSelect 2.0.39 was used to filter and group the data files derived from SORCERER-SEQUEST. The filter thresholds were set to XCorr 2+, 3+, 4+: > 2.5, > 3.0, > 3.5 and DeltaCN > 0.08. For protein identification, two peptides were required with estimated false-discovery rates (FDR) < 1%. For phosphopeptide identification, only unique peptide was required with FDR <2%, whereas DeltaCN should be adjusted to >0.125 if the FDR for phosphopeptide assignments was >2%. Furthermore, additional filtering was used to remove those indubitably false phosphopeptides, including sequences that contained both heavy and light isotopic variants of arginine and lysine. Finally, the FDR fall within 1%.

Dataset Information

Determination of Phosphorylation Sites in the DivIVA Protein of Streptomyces coelicolor by Targeted LC-MS/MS

Publications

Determination of phosphorylation sites in the DivIVA cytoskeletal protein of Streptomyces coelicolor by targeted LC-MS/MS.

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