proteoform profile mapping of the human serum complement component c9 reveals unexpected new features of n o and c glycosylation

Analytical Chemistry is published by the American Chemical Society.. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04527 • Publication Date Web: 21 Feb 2017 Downloaded fr

Analytical Chemistry is published by the American Chemical Society 1155 Sixteenth Street N.W., Washington, DC 20036

C9 reveals unexpected new features of N-, O- and C-glycosylation

Vojtech Franc, Yang Yang, and Albert J.R Heck

Anal Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04527 • Publication Date (Web): 21 Feb 2017

Downloaded from http://pubs.acs.org on February 22, 2017

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Proteoform profile mapping of the human serum Complement component C9

density lipoprotein receptor class A repeat; MAC, membrane attack complex; MS, mass

Most proteins in human blood plasma are decorated by a plethora of PTMs, particularly

process and thus also in the assembly of MAC25, the role of the here identified

R3 50 µm particles28, dried and dissolved in 40 uL of 0.1% FA prior liquid chromatography

mass tolerance, 20 ppm; fixed modification, Cys carbamidomethyl; variable modification:

We started our investigation by first acquiring high-resolution native ESI-MS spectra of the

native MS spectrum of C9 exposed another lower abundant ion series in the higher mass

Denaturation of C9 through the addition of 1% formic acid resulted in a release of Ca2+ and

with up two O-glycans; however, their precise location could not be determined, as the

the both glycan units, HexNAc and Hex The most abundant form (52%) form represents

Although the presence of O-glycans on C9 was proposed more than 20 years ago21, no direct

the fact that such analyses happen under denaturing conditions Our data indicate that C9 can

19 Biesecker, G.; Müller-Eberhard, H J J Immunol 1980, 124, 1291–1296

44 Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y Nat Methods 2015, 12, 7-8

proteoform with m/z of 4,435.28 was deduced as described in Figure 2 Mass differences

Figure 3 Low energy HCD MS/MS (a) and EThcD MS/MS (b) spectra of the peptide harboring the novel,

non-canonical N-glycosylation site at N215 83x75mm (300 x 300 DPI)