TY - JOUR

T1 - An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

AU - Hägglund, Per

AU - Matthiesen, Rune

AU - Elortza, Felix

AU - Højrup, Peter

AU - Roepstorff, Peter

AU - Jensen, Ole Nørregaard

AU - Bunkenborg, Jakob

PY - 2007

Y1 - 2007

N2 - 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 H2 18O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β-N-acetylglusaminidases (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 (β-galactosidase, neuraminidase and N-acetyl-β-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.

AB - 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 H2 18O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β-N-acetylglusaminidases (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 (β-galactosidase, neuraminidase and N-acetyl-β-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.

KW - Diagnostic ions

KW - Fucosylation

KW - Glycosylation

KW - HILIC

KW - Mass spectrometry

KW - Plasma proteins

KW - Post-translational modifications

KW - Proteomics

U2 - 10.1021/pr0700605

DO - 10.1021/pr0700605

M3 - Journal article

C2 - 17636988

AN - SCOPUS:34548186744

VL - 6

SP - 3021

EP - 3031

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 8

ER -