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PXD000188

PXD000188 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleProfiling protein kinases and other ATP binding proteins in Arabidopsis using acyl-ATP probes
DescriptionMany protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using Acyl-ATP (AcATP) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Protein extracts were labelled with NHS-LC-Biotin, purified over neutravidin and eluted with formic acid. Eluted proteins were separated by 1-D SDS Gel and digested with Trypsin. Peptide mixtures were analysed by LC-MS/MS on an LTQ Velos. Spectra were searched using Mascot 2.3 using a fixed modification of cysteine (57.02 Da for carbamidomethyl), and variable modifications of methionine (15.99 Da for oxidation) and lysine (339.16 and 355.16 Da for BHAc and OBHAc labeling, respectively). A mass tolerance was set to 0.3 Da for the precursor ions and 0.4 Da for fragment ions. Up to two mis-cleavages for trypsin were permitted since the labeling would prevent cleavage at the labeled lysine. The MS2 spectra were searched against the TAIR10_pep_2012 (version November 2012) containing 35386 proteins of Arabidopsis thaliana, supplemented with a small database with 1095 artefact proteins and a reversed decoy database of the same proteins (totals 72962 entries). An oxidized version of the modifier was observed. Alternatively Arabidopsis leaf proteome was labeled with desthiobiotin-acyl ATP and digested with trypsin. Labeled peptides were purified on streptavidin matrix and sequenced by LC-MS/MS on an LTQ linear ion trap. Here the MS2 spectra were searched using SEQUEST against the TAIR9_pep_20090619 database (version June 2009) containing 32,769 proteins of Arabidopsis thaliana. MS2 searches included fixed iodoacetamide modification of cysteine (57 Da for alkylation), and variable modifications of methionine (16 Da for oxidation) and lysine (196 Da for DBAcATP labeling). Up to three mis-cleavages with trypsin were allowed and non-tryptic or half-tryptic peptides were excluded. A mass tolerance was set to 3 Da for the precursor ions and 1 Da for fragment ions.
HostingRepositoryPRIDE
AnnounceDate2013-06-19
AnnouncementXMLSubmission_2013-06-19_03:20:17.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterThomas Colby
SpeciesList scientific name: Arabidopsis thaliana (Mouse-ear cress); NCBI TaxID: 3702;
ModificationListiodoacetamide derivatized residue; monohydroxylated residue; NHS-LC-Biotin
InstrumentLTQ
Dataset History
RevisionDatetimeStatusChangeLog Entry
02013-03-21 03:03:26ID requested
12013-06-19 03:18:11announced
12014-07-25 01:00:35announced
22013-06-19 03:20:17announcedAdd reference
Publication List
Villamor JG, Kaschani F, Colby T, Oeljeklaus J, Zhao D, Kaiser M, Patricelli MP, van der Hoorn RA, Profiling protein kinases and other ATP binding proteins in Arabidopsis using Acyl-ATP probes. Mol Cell Proteomics, 12(9):2481-96(2013) [pubmed]
Keyword List
submitter keyword: arabidopsis, activity-based protein profiling, ABPP, kinases, LC-MSMS
Contact List
Thomas Colby
contact affiliationProteomics Group
contact emailcolby@mpipz.mpg.de
Full Dataset Link List
Dataset FTP location
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