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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 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 five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095.
Highlights
We describe the use of targeted LC−MS/MS and different software tools to determine the phosphorylation sites in the Streptomyces coelicolor cytoskeletal element DivIVA
Previous MALDI-TOF analysis of DivIVA immunoprecipitated from S. coelicolor following bacitracin treatment showed that a large 7.2 kDa tryptic peptide (TPATASLPPSPAPSMAPAGA SAPSYGGNQSMGGGPGQSGP SYGGQQQMSPAMTQPMAPVR PQGPSPMGQAPSPMR) that contains most of the C-terminal region of DivIVA was multiply phosphorylated.[1]
Two runs were performed with different tryptic digests of a DivIVA protein preparation using a data-dependent mode of acquisition including dynamic exclusion and chromatographic peak detection
Summary
We describe the use of targeted LC−MS/MS and different software tools to determine the phosphorylation sites in the Streptomyces coelicolor cytoskeletal element DivIVA. Previous MALDI-TOF analysis of DivIVA immunoprecipitated from S. coelicolor following bacitracin treatment showed that a large 7.2 kDa tryptic peptide (TPATASLPPSPAPSMAPAGA SAPSYGGNQSMGGGPGQSGP SYGGQQQMSPAMTQPMAPVR PQGPSPMGQAPSPMR) that contains most of the C-terminal region of DivIVA (see Figure 4) was multiply phosphorylated.[1] The identification of the phosphorylation sites is necessary so that they can be mutagenised to unphosphorylatable (Ala) or phosphomimetic (Asp) residues to analyze the effect of phosphorylation on DivIVA activity further To this end, two additional tryptic cleavage sites were introduced into the 7.2 kDa fragment by mutagenesis. Phosphopeptide isomers are missed during data-dependent LC−MS/MS acquisitions if they elute closely or together because the mass spectrometer is usually set up to implement a dynamic exclusion window to avoid repetitive fragmentation of peptides. The mass spectrometry proteomics data (including the Perl script for plotting of fragments) have been deposited with the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository[7] with the data set identifier PXD000095 and DOI 10.6019/ PXD000095 (PRIDE accessions 29619−29621)
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