Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells.

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Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by 1D SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. GO analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD001081 (http://proteomecentral.proteomexchange.org/dataset/PXD001081).", "claims": [{"id": "claim-target-result", "statement": "The study used one-dimensional SDS-PAGE coupled with HPLC-MS/MS to profile rough ER purified from MIN6 cells, identified 1,467 proteins, and deposited the mass-spectrometry proteomics data in ProteomeXchange via PRIDE (PXD001081; DOI 10.6019/PXD001081).", "kind": "quantitative", "target": {"value": 95.0, "unit": "per cent", "tolerance": null, "source": "A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments."}, "evidence": [{"asset": "source:pmid:25546123", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "In each experiment, the ER pellets were solubilized and analyzed by one dimensional SDS-PAGE coupled with HPLC-MS/MS."}, {"asset": "source:pmid:25546123", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments."}, {"asset": "source:pmid:25546123", "locator": "sections[7] \"p\" paragraph 1", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [21] via the PRIDE partner repository with the dataset identifier PXD001081 and DOI 10.6019/PXD001081"}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "The study used one-dimensional SDS-PAGE coupled with HPLC-MS/MS to profile rough ER purified from MIN6 cells, identified 1,467 proteins, and deposited the mass-spectrometry proteomics data in ProteomeXchange via PRIDE (PXD001081; DOI 10.6019/PXD001081).", "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:25546123"], "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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