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PXD000560-2

PXD000560 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleChasing protein arginine phosphorylation: development of a selective enrichment method using a trapping mutant phosphatase
DescriptionProof-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.
HostingRepositoryPRIDE
AnnounceDate2014-07-24
AnnouncementXMLSubmission_2014-07-24_05:18:03.xml
DigitalObjectIdentifierhttps://dx.doi.org/10.6019/PXD000560
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterDebora Broch Trentini
SpeciesList scientific name: Bacteria; NCBI TaxID: 2; scientific name: Bacillus subtilis subsp. subtilis str. 168; NCBI TaxID: 224308;
ModificationListiodoacetamide derivatized residue; phosphorylated residue; L-methionine sulfoxide
InstrumentLTQ Orbitrap Velos
Dataset History
RevisionDatetimeStatusChangeLog Entry
02013-11-01 01:51:41ID requested
12014-05-19 07:27:26announced
22014-07-24 05:18:04announcedUpdated project metadata.
Publication List
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]
Keyword List
ProteomeXchange project tag: PRIME-XS Project
curator keyword: Biological, Technical
submitter keyword: arginie phosphorylation, trapping mutant phosphatase
Contact List
Karl Mechtler
contact affiliationThe Research Institute of Molecular Pathology (IMP) Institute of Molecular Biotechnology (IMBA)
contact emailmechtler@imp.ac.at
lab head
Debora Broch Trentini
contact affiliationMass spectrometry
contact emailbroch@imp.ac.at
dataset submitter
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Dataset FTP location
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PRIDE project URI
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