title: Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation.
abstract: The human complement C9 protein (∼65 kDa) is a member of the complement pathway. It plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on the cellular surface of pathogenic bacteria. Here, we charted in detail the structural microheterogeneity of C9 purified from human blood serum, using an integrative workflow combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The proteoform profile of C9 was acquired by high-resolution native mass spectrometry, which revealed the co-occurrence of ∼50 distinct mass spectrometry (MS) signals. Subsequent peptide-centric analysis, through proteolytic digestion of C9 and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) measurements of the resulting peptide mixtures, provided site-specific quantitative profiles of three different types of C9 glycosylation and validation of the native MS data. Our study provides a detailed specification, validation, and quantification of 15 co-occurring C9 proteoforms and the first direct experimental evidence of O-linked glycans in the N-terminal region. Additionally, next to the two known glycosylation sites, a third novel, albeit low abundant, N-glycosylation site on C9 is identified, which surprisingly does not possess the canonical N-glycosylation sequence N-X-S/T. Our data also reveal a binding of up to two Ca2+ions to C9. Mapping all detected and validated sites of modifications on a structural model of C9, as present in the MAC, hints at their putative roles in pore formation or receptor interactions. The applied methods herein represent a powerful tool for the unbiased in-depth analysis of plasma proteins and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of the pathophysiological state of the patients.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Vojtech
authors.initials: V
authors.lastname: Franc
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Albert J R
authors.identifier: 0000-0002-2405-4404
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D002853:Chromatography, Liquid; D003186:Complement C9 / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D040901:Proteomics
chemical_list: D003186:Complement C9