An Enzymatic Deglycosylation Scheme Enabling Identification of Core Fucosylated N-Glycans and O-Glycosylation Site Mapping of Human Plasma Proteins Per Ha 1 gglund, ‡ Rune Matthiesen, † Felix Elortza, † Peter Højrup, Peter Roepstorff, Ole Nørregaard Jensen, and Jakob Bunkenborg* Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Received January 31, 2007 Global proteome analysis of protein glycosylation is a major challenge due to the inherent heteroge- neous 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 H 2 18 O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18 O into the formed aspartic acid residue. (2) Digestion with two endo--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 (-galactosidase, neuraminidase and N-acetyl--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. Keywords: proteomics • post-translational modifications • mass spectrometry • HILIC • glycosylation • diagnostic ions • plasma proteins • fucosylation Introduction Rapid developments in mass spectrometry have provided new opportunities for systematic analysis of post-translational modifications (PTMs) on a proteome scale. 1,2 This development has led to an augmented understanding of the importance of PTMs in various cellular processes and diseases. 3 In the context of PTM analysis, glycosylation is a particular challenge. Gly- cosylation is one of the most common types of PTMs, compris- ing a class of structurally diverse modifications, including N-, O-, and C-linked glycans with carbohydrate moieties ranging from monosaccharides to large branched oligosaccharide chains composed of 7-40 monomers attached to the polypep- tide chain. N-linked glycans are large structures containing a conserved trimannosyl chitobiose pentasaccharide core at- tached to asparagine residues in an NX(S/T/C) motif where X can be any amino acid except proline. 4,5 O-linked glycans are most often attached to the hydroxyl groups of serine or threonine residues. They lack a defined attachment motif or common core structure and are, in general, more structurally diverse than N-glycans. 6 C-glycosylation is a single mannose residue linked to tryptophan residues through a carbon-carbon bond to the indole moiety. 7 Because of the structural complexity of glycans, compre- hensive structural analysis of protein glycosylation is predomi- nantly applied to purified proteins. Proteome-scale glycosyl- ation analysis is typically focused on attachment-site analysis. In glycosylation-directed proteomics protocols, methods for enrichment of glycoproteins and/or glycopeptides include lectin affinity chromatography, peroxidase oxidation followed by hydrazide coupling, hydrophilic interaction chromatography (HILIC), boronic acid chromatography, or glycosylation-specific antibodies. 8-12 Identification of glycan attachment points in peptide chains is then facilitated by complete or partial removal of the glycan chain, either by chemical or enzymatic cleav- age, 13-15 or by fragmentation in the mass spectrometer. 16 There are several analytical benefits of removing the major part of * Author to whom correspondence should be addressed. E-mail: bunkenborg@bmb.sdu.dk. † Present address: Cooperative Research Centre on Biosciences (CIC; bioGUNE), Technology park of Bizkaia, 801 A Building, 48160 Derio, Spain. ‡ Present address: Enzyme and Protein Chemistry, Biocentrum DTU 224- 124, Technical University of Denmark, DK-2800 Kgs, Lyngby, Denmark. 10.1021/pr0700605 CCC: $37.00  2007 American Chemical Society Journal of Proteome Research 2007, 6, 3021-3031 3021 Published on Web 07/18/2007