PXD000560-2

PXD000560 is an original dataset announced via ProteomeXchange.

Title	Chasing protein arginine phosphorylation: development of a selective enrichment method using a trapping mutant phosphatase
Description	Proof-of-principle pull-down experiments using a YwlE trapping mutant phosphatase to selective capture arginine phosphorylated proteins. Phosphoarginine is an acid labile protein modification that was demonstrated to exist in bacteria and probably also in higher eukaryotes. Due to the high abundance of Ser, Thr, and Tyr modification, specific enrichment of this type of phosphorylation is required to detect this modification in complex samples and distinguish them from other protein phosphorylations. For this purpose, the phosphoarginine-specific phosphatase YwlE (G.stearothermophilus) was modified by introduction of mutations in catalytically-active amino acids C9 and D118, as well as the surface residue F39. The efficiency of these mutants to enrich arginine phosphorylated proteins from B. subtilis cell extracts was tested in comparison to a shotgun phosphoproteomics approach. Phosphatase trapping mutants were expressed in E.coli with a C-terminal histidine-tag. B.subtilis ywle cultures were grown in LB medium and heat shocked to induce arginine phosphorylation by the protein arginine kinase McsB. Cells were lysed under native conditions and incubated with the trapping mutant. Phosphorylated proteins were enriched by Ni2+/NTA chromatography to isolate the trapping mutant and bound substrates. Sample were prepared for bottom up proteomic analysis by reduction of disulfides, subsequent alkylation of free cysteines and tryptic digestion on the beads. Phosphopeptides were enriched from the resulting peptide mixtures using an optimized TiO2-based enrichment protocol and subsequently submitted to LC-MS/MS analysis.
HostingRepository	PRIDE
AnnounceDate	2014-07-24
AnnouncementXML	Submission_2014-07-24_05:18:03.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD000560
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Debora Broch Trentini
SpeciesList	scientific name: Bacteria; NCBI TaxID: 2; scientific name: Bacillus subtilis subsp. subtilis str. 168; NCBI TaxID: 224308;
ModificationList	iodoacetamide derivatized residue; phosphorylated residue; L-methionine sulfoxide
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-11-01 01:51:41	ID requested
1	2014-05-19 07:27:26	announced
⏵ 2	2014-07-24 05:18:04	announced	Updated project metadata.

Trentini DB, Fuhrmann J, Mechtler K, Clausen T, Chasing Phosphoarginine Proteins: Development of a Selective Enrichment Method Using a Phosphatase Trap. Mol Cell Proteomics, 13(8):1953-64(2014) [pubmed]

ProteomeXchange project tag: PRIME-XS Project

curator keyword: Biological, Technical

submitter keyword: arginie phosphorylation, trapping mutant phosphatase

Karl Mechtler
contact affiliation	The Research Institute of Molecular Pathology (IMP) Institute of Molecular Biotechnology (IMBA)
contact email	mechtler@imp.ac.at
lab head
Debora Broch Trentini
contact affiliation	Mass spectrometry
contact email	broch@imp.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000560
     1. Label: PRIDE project
     2. Name: Chasing protein arginine phosphorylation: development of a selective enrichment method using a trapping mutant phosphatase