Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

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One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180).", "claims": [{"id": "claim-target-result", "statement": "Using the refined TiO2 enrichment method and 9 h LC-MS/MS, the authors analyzed 1 mg of digested AD brain lysate and identified 5,243 phosphopeptides containing 3,715 non-redundant phosphosites on 1,455 proteins, including 31 tau phosphosites.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:25307156", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5,243 phosphopeptides containing 3,715 non-redundant phosphosites on 1,455 proteins, including 31 phosphosites on the tau protein."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Using the refined TiO2 enrichment method and 9 h LC-MS/MS, the authors analyzed 1 mg of digested AD brain lysate and identified 5,243 phosphopeptides containing 3,715 non-redundant phosphosites on 1,455 proteins, including 31 tau phosphosites.", "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:25307156"], "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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