An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins.
Global proteome analysis of protein glycosylation is a major challenge due to the inherent heterogeneous and diverse nature of this post-translational modification. It is therefore common to enzymatically remove glycans attached to protein or peptide chains prior to mass spectrometric analysis, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide-N-glycosidase (PNGase) digestion, with concomitant deamidation of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-beta-N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond between the two N-acetylglucosamine (GlcNAc) residues in the conserved N-glycan core structure, leaving single GlcNAc residues with putative fucosyl side chains attached to the peptide. To enable digestion of complex and hybrid type N-glycans, a number of exoglycosidases (beta-galactosidase, neuraminidase and N-acetyl-beta-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides were also identified with a single N-acetylhexosamine attached, arguably due to partial deglycosylation of O-glycan structures by the exoglycosidases used together with Endo D/H.
Hägglund
Per
P
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Matthiesen
Rune
R
Elortza
Felix
F
Højrup
Peter
P
Roepstorff
Peter
P
Jensen
Ole Nørregaard
ON
Bunkenborg
Jakob
J
D001216:Asparagine / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D010103:Oxygen Isotopes / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry
D001798:Blood Proteins; D010103:Oxygen Isotopes; D011134:Polysaccharides; D001216:Asparagine; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase