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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 J+Am+Soc+Mass+Spectrom
2018 ; 29
(6
): 1099-1110
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Comprehensive Proteoform Characterization of Plasma Complement Component C8??? by
Hybrid Mass Spectrometry Approaches
#MMPMID29532326
Franc V
; Zhu J
; Heck AJR
J Am Soc Mass Spectrom
2018[Jun]; 29
(6
): 1099-1110
PMID29532326
show ga
The human complement hetero-trimeric C8??? (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 C8? and
C8?. Lastly, our data contain the first experimental evidence of O-linked glycans
located on C8?. 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 Abstract
?.