Glycosylation sites separation

B. Kuster and M. Mann, 0-Labeling of A-glycosylation sites to improve the identification of gel-separated glycoproteins nsing peptide mass mapping and database searching, A aZ. Chem. 71 (1999), 1431-1440. 

Table 1 summarizes the carbohydrate structural data deduced from the three detection methodologies, MALDI-TOF, ES-MS, and high pH anion exchange chromatography. The N-glycosylation site proved to be occupied with several structures. The glycopeptides were partially separated as five peptides, at retention times of 19.4, 20.3, 20.7, 20.9, and 21.3 minutes, and all of these glycopeptides contained the same amino acid sequence, N-terminal amino acids 1-17. 

The determination of the glycosylation position is also possible based on lH acetylation shifts after complete acetylation of the metabolites. Protons at the acetoxylated carbons resonate about 1 ppm downfield in comparison with the non-derivatized metabolite, whereas those at the glycosylation sites are only slightly shifted. For example, the non-separated mixture of the two metabolites 2-0-p-D-glucopyranosyl-3,24-diepicastasterone (98) and 3-O-p-D-glucopyranosyl-3,24-diepicastasterone (99) show after acetylation in the - H COSY spectrum two sets of H-2p/H-3a signals at 8 4.92/3.61 (major component) and 3.76/4.74 (minor component), respec-... 

Figure 4-2 Schematic of Fet3p. The predicted Fet3p is 636 amino acids in length and possesses two hydrophobic regions at the N and C-termini. The protein also contains 12 separate potential N-glycosylation sites. In addition, Fet3p contains three domains which correspond to multicopper oxidase motifs.

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