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. 2017 Sep 1;16(9):3348-3362.
doi: 10.1021/acs.jproteome.7b00359. Epub 2017 Jul 27.

An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry

Ebtesam A Gashash  1   2 , Arya Aloor  1 , Dong Li  3 , He Zhu  1 , Xiao-Qian Xu  4 , Cong Xiao  1 , Junping Zhang  5 , Aishwarya Parameswaran  1 , Jing Song  1 , Cheng Ma  1 , Weidong Xiao  5 , Peng George Wang  1
Affiliations

An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry

Ebtesam A Gashash et al. J Proteome Res. .

Abstract

Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms. An RP-LC-MS/MS system functionalized with CID and HCD tandem mass was utilized to analyze VWF. N-Glycosite occupancy varied along the protein backbone chain. Out of 257 HCD spectra, 181 characterized glycoforms were specified as either N- or O-glycosites. Sequential cleavage of glycosidic bonds along with Human Database mass matching have confirmed the glycoform structures. A total of 173 glycoforms represented most commonly biantennary and infrequently tri- and tetra-antennary N-glycans beside high mannose, hybrid, ABH antigen-terminated, and sulfated N-glycans. Many glycoforms were common across all N-sites. Noteworthy, previously unreported N-glycosites within domain D'(TIL'-E') showed glycosylation. Moreover, sialylated core 1 and core 2 O-glycans were detected on 2298T. Given subtle characterization of site-specific glycoforms, we can attain a profound understanding of the biological roles of VWF as well as facilitate the production of VWF-based therapeutics.

Keywords: CID; HCD; N-glycan; O-glycan; VWF; glycosylation; mass spectrometry; microheterogeneity; occupancy; plasma von Willebrand Factor.

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Figures

Figure 1.
Figure 1.
Proteomic analysis of therapeutic VWF by RPLC–ESI–CID–MS. The upper depiction represents the full length VWF including distinct domains, binding sites, and N- and O-glycosites. The lower part represents the coverage of (A) full length VWF (2813 AAs) and (B) mature VWF (2050 AAs).
Figure 2.
Figure 2.
N-Glycosite occupancy of VWF analyzed by RP LC–ESI–CID–MS. The glycoprotein was tryptic digested and then treated with PNGase F in the presence of 18O-water. The asparagine residues are converted into aspartic acid with a mass increment of +2.988 Da, signaling pre-N-glycosylation. (green triangle) N-glycosites, (open blue circle) nonscreened sites, (green circle) occupied, and (blue circle) unoccupied.
Figure 3.
Figure 3.
Extracted ion chromatogram (XIC), MS1, and HCD-MS2 spectra of (KVN2290CTTQPCPTAK) glycopeptide derived from VWF. Y1n+ ([peptide + GlcNAc]n+), Yon+([peptide]n+). (blue square) N-Acetylglucosamine (GlcNAc), (yellow square) N-acetylgalactosamine (GalNAc), (green circle) mannose (Man), (yellow circle) galactose (Gal), (purple diamond) N-acetylneuraminic acid (Neu5Ac), and (red triangle) fucose.
Figure 4.
Figure 4.
Characterized HCD spectra of intact VWF glycopeptides carrying 820NRC or 847NTC N-glycosites. Y1n+ ([peptide + GlcNAc]n+), Yon+([peptide]n); y- and b-ions are peptide backbone fragments. (blue square) N-Acetylglucosamine (GlcNAc), (yellow square) N-acetylgalactosamine (GalNAc), (green circle) mannose (Man), (yellow circle) galactose (Gal), (purple diamond) N-acetylneuraminic acid (Neu5Ac), and (red triangle) fucose.
Figure 5.
Figure 5.
Microheterogeneity and relative abundances of N- and O-glycoforms attached to specific glycosites of VWF. (blue square) N-Acetylglucosamine (GlcNAc), (yellow square) N-acetylgalactosamine (GalNAc), (green circle) mannose (Man), (yellow circle) galactose (Gal), (purple diamond) N-acetylneuraminic acid (Neu5Ac), and (red triangle) fucose.
Figure 6.
Figure 6.
Characterized HCD spectra of di- and trisialylated core 1 and monosialylated core 2 glycoforms attached to the 2298T O-glycosite of VWF. Y1n+ ([peptide + GalNAc]n+), Yon+([peptide]n+); y- and b-ions are peptide backbone fragments. (blue square) N-Acetylglucosamine (GlcNAc), (yellow square) N-acetylgalactosamine (GalNAc), (yellow circle) galactose (Gal), and (purple diamond) N-acetylneuraminic acid (Neu5Ac).
Figure 7.
Figure 7.
General characterization of plasma VWF N-glycome (61 compositions). (A) Total relative abundance (TRA) (bars; left side) and site-specific relative abundance (SRA (heat map; right side) of N-glycan compositions). (B) Distribution of N-glycan complexity (the outer circle represents the percentage of sulfated glycans of each type). (blue square) N-Acetylglucosamine (GlcNAc, N), (yellow square) N-acetylgalactosamine (GalNAc, N), (green circle) mannose (Man, H), (yellow circle) galactose (Gal, H), (purple diamond) N-acetylneuraminic acid (Neu5Ac, S), (red triangle) fucose (Fuc, F), and sulfate groups (s).
Figure 8.
Figure 8.
Classification of 173 N-glycoforms specified into VWF glycosites. (A) Relative abundance of VWF N-glycoform types. (B) Number and relative abundance of ABH antigens containing N-glycoforms. (blue square) N-Acetylglucosamine (GlcNAc), (yellow square) N-acetylgalactosamine (GalNAc), (green circle) mannose (Man), (yellow circle) galactose (Gal), (purple diamond) N-acetylneuraminic acid (Neu5Ac), and (red triangle) fucose.
Scheme 1.
Scheme 1.. Schematic Depiction of the Overall Workflow for Proteomic/Glycoproteomic Analysis of Plasma VWF by RP–LC–ESI–MS/MSa
aY1n+ ([peptide + GlcNAc]n+), Yon+([peptide]n+); (blue square) N-acetylglucosamine (GlcNAc), (yellow square) N-acetylgalactosamine (GalNAc), (green circle) mannose (Man), (yellow circle) galactose (Gal), and (purple diamond) N-acetylneuraminic acid (Neu5Ac).
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