Comparative and optimized dabsyl-amino acid analysis of synthetic phosphopeptides and glycopeptides.
The optimal conditions for amino acid analysis of phosphopeptides and N-acetylglucosamine- and N-acetylgalactosamine-containing glycopeptides were investigated by dabsyl-Cl derivatization and reversed-phase high-performance liquid chromatographic separation. By comparing the chromatographic behaviour of the dabsylated phosphoamino acids and dabsylated aminosugars on three different columns, it appears that the mechanism of binding to the column is different for the two modified dabsyl derivatives. The acid sensitivities of sugar and phosphate groups were also investigated. We found that while the optimal hydrolysis conditions for phosphopeptide analysis are peptide sequence-dependent, there is generally an applicable condition for the highest recovery of glycopeptides. A 1-h gas-phase hydrolysis time seems to be appropriate for the majority of glycopeptides and 1.5 h is suitable for the majority of phosphopeptides. The analysis was extended to the successful verification of the presence of the N-acetylglucosamine and the N-acetylgalactosamine moieties when these sugars were incorporated as parts of a disaccharide side chain of glycopeptides.
Gorbics
L
L
Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104-4268.
Urge
L
L
Otvos
L
L
D000116:Acetylgalactosamine / Q000032:analysis; D000117:Acetylglucosamine / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004187:Disaccharides / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives*
D000596:Amino Acids; D004187:Disaccharides; D006023:Glycoproteins; D007202:Indicators and Reagents; D010748:Phosphopeptides; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D004124:p-Dimethylaminoazobenzene; D000116:Acetylgalactosamine; D000117:Acetylglucosamine