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Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation.
Franc, Vojtech; Yang, Yang; Heck, Albert J R.
Afiliação
  • Franc V; 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.
  • Yang Y; Netherlands Proteomics Center , Padualaan 8, 3584 CH Utrecht, The Netherlands.
  • Heck AJ; 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.
Anal Chem ; 89(6): 3483-3491, 2017 03 21.
Article em En | MEDLINE | ID: mdl-28221766
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Complemento C9 / Proteômica Limite: Humans Idioma: En Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Complemento C9 / Proteômica Limite: Humans Idioma: En Ano de publicação: 2017 Tipo de documento: Article