Analysis of pancreatic extracellular matrix protein post-translational modifications via electrostatic repulsion-hydrophilic interaction chromatography coupled with mass spectrometry.

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A better understanding of the pancreas proteome and its dynamic post-translational modifications (PTMs) is necessary to engineer higher fidelity tissue analogues for use in transplantation. The extracellular matrix (ECM) has major roles in binding and signaling essential to the viability of insulin-producing islets of Langerhans. To characterize PTMs in the pancreas, native and decellularized tissues from four donors were analyzed. N-Glycosylated and phosphorylated peptides were simultaneously enriched via electrostatic repulsion-hydrophilic interaction chromatography and analyzed with mass spectrometry, maximizing PTM information from one workflow. A modified surfactant and chaotropic agent assisted sequential extraction/on-pellet digestion was used to maximize solubility of the ECM. The analysis resulted in the confident identification of 3650 proteins, including 517 N-glycoproteins and 148 phosphoproteins. We identified 214 ECM proteins, of which 99 were N-glycosylated, 18 were phosphorylated, and 9 were found to have both modifications. Collagens, a major component of the ECM, were the most highly glycosylated of the ECM proteins and several were also heavily phosphorylated, raising the possibility of structural and thus functional changes resulting from these modifications. To our knowledge, this work represents the first characterization of PTMs in pancreatic ECM proteins. This work provides a basal profile of PTMs in the healthy human pancreatic ECM, laying the foundation for future investigations to determine disease-specific changes such as in diabetes and pancreatic cancer, and potentially helping to guide the development of tissue replacement constructs. Data are available via ProteomeXchange with identifier PXD025048.", "claims": [{"id": "claim-method", "statement": "Statistical significance was determined using Fischer’s exact test with false discovery rate correction.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34318855", "locator": "sections[9] \"UPLC-MS/MS and data analysis:\" paragraph 4", "quote": "Statistical significance was determined using Fischer’s exact test with false discovery rate correction."}], "state": "complete"}, {"id": "claim-data-access", "statement": "Data are available via ProteomeXchange with identifier PXD025048.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34318855", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data are available via ProteomeXchange with identifier PXD025048."}], "state": "complete"}, {"id": "claim-target-result", "statement": "The analysis resulted in the confident identification of 3,650 proteins, including 517 N-glycoproteins and 148 phosphoproteins.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:34318855", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "The analysis resulted in the confident identification of 3,650 proteins, including 517 N-glycoproteins and 148 phosphoproteins."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "The analysis resulted in the confident identification of 3,650 proteins, including 517 N-glycoproteins and 148 phosphoproteins.", "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:34318855"], "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": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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