Unusual Fragmentation Pathways in Collagen Glycopeptides

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)(n). Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides, i.e. in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed. The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways – amide bond and glycosidic bond cleavage – are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e. Arg, Lys, HyK and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations, to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides

Characterization of Site-Specific N-Glycosylation

Even if a consensus sequence has been identified for a posttranslational modification, the presence of such a sequence motif only indicates the possibility, not the certainty that the modification actually occurs. Proteins can be glycosylated on certain amino acid side chains, and these modifications are designated as C-, N-, and O-glycosylation. C-mannosylation occurs on Trp residues within a relatively loosely defined consensus motif. N-glycosylated species are modified at Asn residues of Asn-Xxx-Ser/Thr/Cys sequons (where Xxx can be any amino acid except proline). N-linked oligosaccharides share a common core structure of GlcNAc2Man3. In addition, an enzyme, peptide N-glycosidase F (PNGase F), removes most of the common N-linked carbohydrates unaltered from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O-glycosylation occurs at Ser, Thr, and Tyr residues, usually in sequence stretches rich in hydroxy-amino acids. O-glycosylation lacks a common core structure. Mammalian proteins have been reported bearing O-linked N-acetylgalactosamine, fucose, glucose, xylose, mannose, and corresponding elongated structures, as well as N-acetylglucosamine. Chemical methods are used to liberate these oligosaccharides because no enzyme would remove all the different O-linked carbohydrates. Characterization of both N- and O-glycosylation is complicated by the fact that the same positions within a population of protein molecules may feature an array of different carbohydrate structures, or remain unmodified. This site-specific heterogeneity may vary by species and tissue, and may also be affected by physiological changes. For addressing site-specific carbohydrate heterogeneity mass spectrometry has become the method of choice. Reversed-phase HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESI-MS/MS) offers the best solution. Using a mass spectrometer as online detector not only assures the analysis of every component eluting (mass mapping), but also at the same time diagnostic carbohydrate ions can be generated by collisional activation that permits the selective and specific detection of glycopeptides. In addition, ESI-compatible alternative MS/MS techniques, electron-capture and electron-transfer dissociation, aid glycopeptide identification as well as modification site assignments