Abstract
The human complement hetero-trimeric C8αβγ (C8) protein assembly (~ 150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~ 20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions. Their native MS analysis revealed even more detailed structural micro-heterogeneity on C8. Subsequent peptide-centric analysis, by proteolytic digestion of C8 and LC-MS/MS, provided site-specific quantitative profiles of different types of C8 glycosylation. Combining all this data provides a detailed specification of co-occurring C8 proteoforms, including experimental evidence on N-glycosylation, C-mannosylation, and O-glycosylation. In addition to the known N-glycosylation sites, two more N-glycosylation sites were detected on C8. Additionally, we elucidated the stoichiometry of all C-mannosylation sites in all the thrombospondin-like (TSP) domains of C8α and C8β. Lastly, our data contain the first experimental evidence of O-linked glycans located on C8γ. Albeit low abundant, these O-glycans are the first PTMs ever detected on this subunit. By placing the observed PTMs in structural models of free C8 and C8 embedded in the MAC, it may be speculated that some of the newly identified modifications may play a role in the MAC formation.Graphical ᅟ
Highlights
I n humans, the complement system forms the first line of defense against microbial infections
The calculated mass was further adjusted by the mass shift induced by the 22 disulfide bonds present in C8 (8 in α, 12 in β, 1 in γ subunit and 1 inter disulfide bond between the α and γ subunits) and by the mass shift − 18.01 Da corresponding to the conversion of the N-terminal Gln on the γ subunit to pyroglutamic acid [27,28,29]
Determining the exact backbone mass allowed us to calculate a mass shift of 5547.97 Da induced by the post-translational modifications (PTMs) on the most abundant peak in the 24+ charge state with m/z 6193.65 (148,623.60– 143,075.63 = 5547.97 Da)
Summary
I n humans, the complement system forms the first line of defense against microbial infections. Highresolution native MS has been combined with peptide-centric proteomics for an in-depth analysis of PTMs on plasma or recombinant proteins [8, 9] We proposed this hybrid MS strategy for the efficient and reliable analysis of biologically important (glyco)proteins, as has been first demonstrated by our analysis of complement component C9 [10]. We report an in-depth analysis of the C8 complement component protein assembly purified from pooled normal human blood plasma using hybrid MS techniques combined with ion exchange chromatography. Our data show that chromatographic separation of C8 from human plasma allows for the isolation and subsequent MS detection of different C8 forms This allows us to obtain a detailed view of the modifications co-occurring on each of the three C8 subunits. Overall our findings bring detailed new insights into the structural composition of C8, an essential factor in complement activation and our immune response
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Journal of The American Society for Mass Spectrometry
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
