Profiling protein kinases and other ATP binding proteins in Arabidopsis using Acyl-ATP probes.

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Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)(1) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.", "claims": [{"id": "claim-target-result", "statement": "The paper plotted spectral counts against expression levels to assess probe specificity, and the underlying data were deposited in ProteomeXchange under identifier PXD000188; the analysis identified 242 AcATP labeling sites in the Arabidopsis proteome.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:23722185", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome."}, {"asset": "source:pmid:23722185", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins."}, {"asset": "source:pmid:23722185", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The data have been deposited in ProteomeXchange with the identifier PXD000188."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "The paper plotted spectral counts against expression levels to assess probe specificity, and the underlying data were deposited in ProteomeXchange under identifier PXD000188; the analysis identified 242 AcATP labeling sites in the Arabidopsis proteome.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:23722185"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. 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