title: Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.
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.
authors.affiliation: From the ‡Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany;
authors.forename: Marcus
authors.initials: M
authors.lastname: Hoffmann
authors.affiliation: §Bruker Daltonik GmbH, 28359 Bremen, Germany;
authors.forename: Kristina
authors.initials: K
authors.lastname: Marx
authors.affiliation: From the ‡Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany; ¶Otto von Guericke University Magdeburg, Chair of Bioprocess Engineering, 39106 Magdeburg, Germany;
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
authors.affiliation: ‖Center for Proteomics and Metabolomics, Department of Rheumatology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands;
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.affiliation: From the ‡Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany; **glyXera GmbH, Leipziger Strasse 44 (Zenit), 39120 Magdeburg, Germany rapp@mpi-magdeburg.mpg.de.
authors.forename: Erdmann
authors.initials: E
authors.lastname: Rapp
mesh_terms: D000209:Acute-Phase Proteins / Q000096:biosynthesis / Q000235:genetics*; D000957:Antigens, Viral, Tumor / Q000097:blood / Q000235:genetics*; D056148:Chromatography, Reverse-Phase; D006020:Glycopeptides / Q000097:blood / Q000235:genetics*; D006031:Glycosylation; D064368:Healthy Volunteers; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D018396:Mucin-1 / Q000097:blood / Q000235:genetics; D011134:Polysaccharides / Q000097:blood / Q000235:genetics*
chemical_list: D000209:Acute-Phase Proteins; D000957:Antigens, Viral, Tumor; D006020:Glycopeptides; D018396:Mucin-1; D011134:Polysaccharides