Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:26598643", "kind": "science-record", "revision": 5, "content_hash": "sha256:497d081d4ba6cfc1cc0c39d4e7e128fc7890814232520212e5e8393935e7ff5f", "created_at": "2026-11-01T15:40:14Z", "updated_at": "2026-11-01T18:47:48Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.", "doi": "10.1074/mcp.M115.053546", "pmid": "26598643", "authors": ["Marcus Hoffmann", "Kristina Marx", "Udo Reichl", "Manfred Wuhrer", "Erdmann Rapp"], "journal": "Molecular & cellular proteomics : MCP", "year": 2016, "abstract": "Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.", "claims": [{"id": "claim-data-access", "statement": "All mass spectrometry raw data as well as (glyco)peptide identifications and spectra annotations have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the MassIVE repository with the dataset identifier PXD002315or MSV000079141, respectively.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:26598643", "locator": "sections[15] \"Analysis of Glycopeptides\" paragraph 5", "quote": "All mass spectrometry raw data as well as (glyco)peptide identifications and spectra annotations have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the MassIVE repository with the dataset identifier PXD002315or MSV000079141, respectively."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": null, "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:26598643"], "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_unresolved", "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PXD002315"], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PXD002315"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": ["step:obtain_source_data blocked on data:accession_unresolved"]}, "data": {"state": "partial", "assets": [{"id": "source:pmid:26598643", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1074/mcp.M115.053546", "sha256": "sha256:faebdaae44b098ebe51f69d0ebafb852b26258a05bd59dd8e80ca846d675e354", "bytes": 77244, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1074/mcp.M115.053546", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC4739677", "source": "europe_pmc_fulltext", "coverage": "whole_body_without_references", "text_hash_scope": "rendered_section_body"}}, {"id": "data:geo:PXD002315", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=PXD002315", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "PXD002315", "version": null, "note": "All mass spectrometry raw data as well as (glyco)peptide identifications and spectra annotations have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the MassIVE repository with the dataset identifier PXD002315or MSV000079141, respectively.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "All mass spectrometry raw data as well as (glyco)peptide identifications and spectra annotations have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the MassIVE repository with the dataset identifier PXD002315or MSV000079141, respectively.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": null, "resolution_checked_at": "2026-10-31T18:35:55Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "data:geo:PXD003270", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=PXD003270", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "PXD003270", "version": null, "note": "All mass spectrometry raw data as well as (glyco)peptide identifications and spectra annotations have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the MassIVE repository with the dataset identifier PXD002315or MSV000079141, respectively.", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "All mass spectrometry raw data as well as (glyco)peptide identifications and spectra annotations have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)via the MassIVE repository with the dataset identifier PXD002315or MSV000079141, respectively.", "access_statement_kind": "data_availability_statement", "sample_accessions": [], "sample_count": null, "group_sizes": null, "group_sizes_basis": null, "platform": null, "taxon": null, "title": null, "resolution_checked_at": "2026-10-31T18:35:55Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}, {"id": "asset:table:pmid:26598643:1", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:4fa80e3150e545c3b70f3e358d37449c619961da1305fcb8eb5c46a0c56b67f0", "bytes": 1138, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[22]/table-wrap[1]#TI", "version": null, "note": "Site-specific O-glycan composition of identified human blood plasma glycoproteins. Glycoproteins are listed with their UniprotKB accession number as well as the number of identified glycopeptides. O-glycosylated sites or regions are indexed with respect to the attached O-glycans (mono- and/or disialylated mucin-type core 1 O-glycans). O-glycosylation sites in bold have been pinpointed within this study. Previously unknown sites und regions are indicated by underlining. Curled brackets mark regions with several possible O-glycosylation sites. Superscript numbers indicate literature references. For every protein the number of registered, previously known, as well as new O-glycosylation sites and regions are given. For underlined proteins, glycosylated as well as non-glycosylated peptides were identified (supplemental Table S1). In addition previously reported plasma concentrations are given. HexNAc (N-acetylhexosamine), Hex (hexose), NeuAc (N-acetylneuraminic acid, sialic acid)", "structure": {"pmcid": "PMC4739677", "section_index": 22, "section_title": "Identification of the Peptide Moiety", "row_count": 0, "column_count": 0}, "table": {"label": "Table I", "caption": "Site-specific O-glycan composition of identified human blood plasma glycoproteins. Glycoproteins are listed with their UniprotKB accession number as well as the number of identified glycopeptides. O-glycosylated sites or regions are indexed with respect to the attached O-glycans (mono- and/or disialylated mucin-type core 1 O-glycans). O-glycosylation sites in bold have been pinpointed within this study. Previously unknown sites und regions are indicated by underlining. Curled brackets mark regions with several possible O-glycosylation sites. Superscript numbers indicate literature references. For every protein the number of registered, previously known, as well as new O-glycosylation sites and regions are given. For underlined proteins, glycosylated as well as non-glycosylated peptides were identified (supplemental Table S1). In addition previously reported plasma concentrations are given. HexNAc (N-acetylhexosamine), Hex (hexose), NeuAc (N-acetylneuraminic acid, sialic acid)", "rows": [], "row_count": 0, "column_count": 0, "numeric_cells": 0, "header_rows": 0, "locator": "section[22]/table-wrap[1]#TI"}}, {"id": "asset:table:pmid:26598643:2", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:b6a8999edea25a8e13e1d469130b3772aafa643d482ed56d691ba16dedb19912", "bytes": 826, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[22]/table-wrap[2]#TII", "version": null, "note": "Detailed overview of all identified human blood plasma O-glycopeptides. For each O-glycopeptide, the corresponding glycoprotein including the UniProtKB accession number, the identified O-glycosylation site(s)/regions as well as the O-glycan composition are given, respectively. Likewise, the LC-MS retention time, the mass of the intact glycopeptide precursor, the measured peptide mass as well as the corresponding error is listed. The peptide identification using CID-MS3 was validated by the MASCOT peptide score and the Biotools score (CID). ETD based determination of the O-glycosylation site(s) was validated by the Biotools score (CID) as well as the NetOGlyc 4.0 score", "structure": {"pmcid": "PMC4739677", "section_index": 22, "section_title": "Identification of the Peptide Moiety", "row_count": 0, "column_count": 0}, "table": {"label": "Table II", "caption": "Detailed overview of all identified human blood plasma O-glycopeptides. For each O-glycopeptide, the corresponding glycoprotein including the UniProtKB accession number, the identified O-glycosylation site(s)/regions as well as the O-glycan composition are given, respectively. Likewise, the LC-MS retention time, the mass of the intact glycopeptide precursor, the measured peptide mass as well as the corresponding error is listed. The peptide identification using CID-MS3 was validated by the MASCOT peptide score and the Biotools score (CID). 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