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Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation

[Image: see text] 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 o...

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Autores principales: Franc, Vojtech, Yang, Yang, Heck, Albert J. R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2017
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362742/
https://www.ncbi.nlm.nih.gov/pubmed/28221766
http://dx.doi.org/10.1021/acs.analchem.6b04527
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author Franc, Vojtech
Yang, Yang
Heck, Albert J. R.
author_facet Franc, Vojtech
Yang, Yang
Heck, Albert J. R.
author_sort Franc, Vojtech
collection PubMed
description [Image: see text] 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 Ca(2+) 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.
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spelling pubmed-53627422017-03-24 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. Anal Chem [Image: see text] 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 Ca(2+) 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. American Chemical Society 2017-02-21 2017-03-21 /pmc/articles/PMC5362742/ /pubmed/28221766 http://dx.doi.org/10.1021/acs.analchem.6b04527 Text en Copyright © 2017 American Chemical Society This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License (http://pubs.acs.org/page/policy/authorchoice_ccbyncnd_termsofuse.html) , which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.
spellingShingle Franc, Vojtech
Yang, Yang
Heck, Albert J. R.
Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation
title Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation
title_full Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation
title_fullStr Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation
title_full_unstemmed Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation
title_short Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation
title_sort proteoform profile mapping of the human serum complement component c9 revealing unexpected new features of n-, o-, and c-glycosylation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362742/
https://www.ncbi.nlm.nih.gov/pubmed/28221766
http://dx.doi.org/10.1021/acs.analchem.6b04527
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