Identification and Characterization of Glycosylated Phenylthiohydantoin Amino Acids
The three major groups of glycosylated phenylthiohydantoin (PTH) derivatives Asn(Sac), Ser(Sac) and Thr(Sac), can be clearly resolved and separated from the other 20 commonly occurring PTH-amino acids using a new 5 mM triethylammonium formate (TEAF) buffer, pH 4.0 with an acetonitrile gradient. The glycosylated amino acids elute early in a 1·5 min “glycosylation window” between 6·5–8 min, while all the other PTH-amino acids elute between 8–15 min. This buffer system was developed principally for its ability to separate all PTH-amino acids and glycoamino acids at low ionic strength. The low buffer concentration is necessary to minimize glucose contamination for monosaccharide analysis of the PTH-glycoamino acids.
We demonstrate that: (a) a TEAF buffer system is compatible with monosaccharide analysis of the PTH-glycoamino acid and, in principle, the volatile nature of the buffer makes it suitable for ionspray mass spectrometric analysis of recovered PTH-glycoamino acids. (b) the “glycosylation window” is important for the detection of site-specific partial glycosylation and for identifying different forms of PTH-glycoamino acids.
KeywordsDictyostelium Discoideum Edman Degradation Monosaccharide Analysis High Performance Anion Exchange Chromatography Terminal Galactose Residue
Unable to display preview. Download preview PDF.
- Gooley, A. A., Classon, B. J. Marschalek, R. and Williams K. L. (1991) Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: The identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation, Biochem. Biophys. Res. Comm. 178: 1194Google Scholar
- Gooley, A. A., Packer, N. H., Pisano, A., Redmond, J. W., Alewood, P. F., Jones, A., Loughnan, M. and Williams, K. L. (1994a) Characterization of N-and 0-linked glycosylation sites using Edman degradation, Techniques in Protein Chemistry VI In PressGoogle Scholar
- Gooley, A. A., Pisano, A., Packer, N. H., Ball, M., Jones, A., Alewood, P. F., Redmond, J. W. and Williams, K. L. (1994b) Characterisation of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation, Glycoconjugate J. In PressGoogle Scholar
- Haynes, P. A., Batley, M. Peach, R. J. and Brennan, S. O. (1992) Characterisation of oligosaccharides from a glycoprotein variant of human serum albumin (albumin Casebrook) using high performance anion exchange chromatography and nuclear magnetic resonance spectroscopy, J. Chromatogr. 581: 187PubMedGoogle Scholar
- Laursen, R. A., Lee, T. T., Dixon, J. D. and Liang, S-P. (1991) Extending the performance of the solid-phase protein sequenater in: Methods in Protein Sequence Analysis, (Eds) Jornall, H., Hoog, J-0. and Gustvasson, A-M., Bikhauser Verlag, Switzerland, pp 47–54Google Scholar
- Pisano, A., Packer, H. N., Redmond, J. W., Williams, K. L. and Gooley, A. A. (1994) Characterisation of linked glycosylation motifs in the glycopeptide domain of bovine x-casein, Glycobiology In Press.Google Scholar
- Strydom, D. J. (1994) On-line separation of phenylthiohydantoin derivatives of hydrophilic modified amino acids during sequencing, J. Chromatogr. 664: 227Google Scholar
- Tarr, G. E. (1986) Manual Edman sequencing system, in: Methods of Protein Microcharacterization, (Ed) Shively, J. E., Humana Press, Clifton, New Jersey, pp 162–163Google Scholar
- Zhou-Chou, T., Slade, M. B., Williams, K. L. and Gooley, A. A. (1994) Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum,(J Biotech.,In Press)Google Scholar