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

Saalbach, Gerhard ; Hempel, Antje M. ; Vigouroux, Marielle ; Flärdh, Klas LU ; Buttner, Mark J. and Naldrett, Michael J. (2013) In Journal of Proteome Research 12(9). p.4187-4192
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... (More)
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 PXD00009S. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Ascore, DivIVA, phosphopeptides, phosphorylation site localization, ScaffoldPTM, targeted LC-MS/MS
in
Journal of Proteome Research
volume
12
issue
9
pages
4187 - 4192
publisher
The American Chemical Society (ACS)
external identifiers
  • wos:000330147800032
  • scopus:84883750854
  • pmid:23905541
ISSN
1535-3893
DOI
10.1021/pr400524d
language
English
LU publication?
yes
id
67d29a2f-63df-4e3f-9192-a4e0fa667f47 (old id 4318794)
date added to LUP
2016-04-01 10:49:11
date last changed
2020-07-01 01:38:03
@article{67d29a2f-63df-4e3f-9192-a4e0fa667f47,
  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 PXD00009S.},
  author       = {Saalbach, Gerhard and Hempel, Antje M. and Vigouroux, Marielle and Flärdh, Klas and Buttner, Mark J. and Naldrett, Michael J.},
  issn         = {1535-3893},
  language     = {eng},
  number       = {9},
  pages        = {4187--4192},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Journal of Proteome Research},
  title        = {Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of Streptomyces coelicolor by Targeted LC-MS/MS},
  url          = {http://dx.doi.org/10.1021/pr400524d},
  doi          = {10.1021/pr400524d},
  volume       = {12},
  year         = {2013},
}