PXD000095

PXD000095 is an original dataset announced via ProteomeXchange.

Title	Determination of Phosphorylation Sites in the DivIVA Protein of Streptomyces coelicolor by Targeted LC-MS/MS
Description	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).
HostingRepository	PRIDE
AnnounceDate	2013-08-05
AnnouncementXML	Submission_2013-08-05_06:31:27.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD000095
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Gerhard Saalbach
SpeciesList	scientific name: Streptomyces coelicolor; NCBI TaxID: 1902;
ModificationList	phosphorylated residue; monohydroxylated residue; neutral loss: 63.998285; iodoacetamide derivatized residue
Instrument	radial ejection linear ion trap

Revision	Datetime	Status	ChangeLog Entry
0	2012-12-11 03:53:49	ID requested
1	2013-08-05 06:09:26	announced
1	2014-07-28 00:26:51	announced
⏵ 2	2013-08-05 06:31:27	announced	Add reference

Saalbach G, Hempel AM, Vigouroux M, Fl, ä, rdh K, Buttner MJ, Naldrett MJ, Determination of phosphorylation sites in the DivIVA cytoskeletal protein of Streptomyces coelicolor by targeted LC-MS/MS. J Proteome Res, 12(9):4187-92(2013) [pubmed]

submitter keyword: Ascore, DivIVA, phosphopeptides, phosphorylation site localization, ScaffoldPTM, targeted LC-MS/MS, Orbitrap

Gerhard Saalbach
contact affiliation	Biological Chemistry
contact email	gerhard.saalbach@jic.ac.uk

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• PRIDE
  1. 29620
     1. InstrumentRef: INSTRUMENT_1
     2. Label: DivIVa
     3. ModificationList: monohydroxylated residue, phosphorylated residue,
     4. Name: DivIVa phosphorylation
     5. PublicationRef: PMID23905541
  2. 29621
     1. InstrumentRef: INSTRUMENT_1
     2. Label: DivIVa
     3. ModificationList: monohydroxylated residue, phosphorylated residue, neutral loss, iodoacetamide derivatized residue,
     4. Name: DivIVa mutant identfication in total S. coelicolor database
     5. PublicationRef: PMID23905541
  3. 29619
     1. InstrumentRef: INSTRUMENT_1
     2. Label: DivIVa
     3. ModificationList: phosphorylated residue,
     4. Name: DivIVa Phosphorylation
     5. PublicationRef: PMID23905541