Characterization of site-specific N-glycosylation.

Even if a consensus sequence has been identified for a post- translational 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 N- and O-glycosylation. N-glycosylated species are modified at Asn residues. There is a consensus sequence for N-glycosylation: AsnXxxSer/Thr/Cys, 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 unaltered most of the common N-linked carbohydrates from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O- glycosylation occurs at Ser or Thr-residues, usually in sequence-stretches rich in hydroxy amino acids, but there has been no consensus sequence determined for this modification. In addition, O-glycosylation lacks a common core structure: mammalian proteins have been reported bearing O-linked N-acetylgalactosamine, fucose, glucose, and corresponding elongated structures, as well as N-acetylglucosamine. Chemical methods are used to liberate these oligosaccharides because no enzyme has been discovered that would cleave all the different O-linked carbohydrates. Characterization of both types of glycosylation is complicated by the fact that the same amino acids within a population of protein molecules may be derivatized with an array of different carbohydrate structures, or remain unmodified. This site-specific heterogeneity may vary by species, tissue, and may be affected by physiological changes, and so on. For addressing site-specific carbohydrate heterogeneity mass spectrometry has become the method of choice. Although matrix-assisted laser desorption ionization mass spectrometry of collected HPLC-fractions has been used successfully for this purpose, reversed phase HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESIMS) offers better resolution. Using a mass spectrometer as on-line detector not only assures the analysis of every component eluting (mass mapping), but at the same time diagnostic carbohydrate ions can be generated by collisional activation in the ion-source that permit the selective detection of glycopeptides.