Determination of phosphorylation sites in the DivIVA cytoskeletal 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, 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 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 plo

SUBMITTER: Saalbach G 

PROVIDER: S-EPMC3787806 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature