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

ORGANISM(S): Streptomyces Coelicolor

SUBMITTER: Gerhard Saalbach

LAB HEAD: Gerhard Saalbach

PROVIDER: PXD000095 | Pride | 2013-08-05

REPOSITORIES: Pride

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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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			Action	DRS
	100308_01.RAW	Raw
	100308_03.RAW	Raw
	100312_01.RAW	Raw
	100315_01.RAW	Raw
	DivIVa_ScaffoldPTM.mzid	Mzid