Synthesis, Evaluation and Application of a Panel of Novel Reagents for Stepwise Degradation of Polypeptides
- 137 Downloads
The Edman degradation (Edman, 1949) has been the most successful, general and widely used technique for the determination of the amino acid sequence of proteins and peptides. As a benefit of this distinction, over the last four decades the method has been refined to a high degree of perfection. Nevertheless, sequencing with phenyl isothiocyanate (PITC)† suffers from a few practical limitations. First, the extinction coefficient of the phenylthiohydantoins (PTH’s) limits sequencing sensitivity. Currently, routine sequencing in most laboratories requires low picomole amounts of sample applied to the sequencer. Second, UV-absorbing products which may co-elute with PTH’s during high performance liquid chromatography (HPLC) separation have a tendency to obscure the specific PTH signals during high sensitivity sequencing. Third, with the exception of select cases (Wettenhall et al, 1991; Meyer et al, 1990, 1991; Aebersold et al, 1991; Gooley et al, 1991; Pisano et al, 1993), modified and unnatural amino acids of known structure are difficult to identify and de-novo characterization of such residues by UV absorbance detection alone is extremely difficult.
KeywordsHigh Performance Liquid Chromatography High Performance Liquid Chromatography Quaternary Amine Amino Acid Derivative Unnatural Amino Acid
high-performance liquid chromatography
electrospray ionization mass spectrometer/metry
tandem mass spectrometer/metry
4-(3 pyridylmethylaminocarboxypropyl)-phenyl isothiocyanate
reverse-phase high-performance liquid chromatography
Unable to display preview. Download preview PDF.
- Bures, E.J., Nika, H., Chow, D.T., Morrison, H.D., Aebersold, R. 1994 Synthesis of the protein-sequencing reagent 4-(3-pyridinylmethylamino-carboxypropyl) phenyl isothiocyanate and characterization of 4-(3-pyridinyl-methylaminocarboxypropyl) phenylthiohydantoins. Anal. Biochem. in press.Google Scholar
- Gooley, A.A., Classon, B.J., Marschalek, R.,, 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. and Biophys. Res. Commun. 178: 1194–1201.CrossRefGoogle Scholar
- Hess, D., Nika, H., Chow, D.T., Bures, E.J., Morrison, H.D., Aebersold, R. 1994 Liquid chromatography-electrospray ionization mass spectrometry of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins. Anal. Biochem. accepted for publication.Google Scholar
- Meyer, H.E., Hoffmann-Posorske, E., Korte, H., Donella-Deana, A, Brunati, A.M., Pinna, L.A., Coull, J., Perich, J., Valerio, R.M., Johns, R.B. 1990 Determination and location of phosphoserine in proteins and peptides by conversion to S-ethylcysteine. Chromatographia 30: 691–695.CrossRefGoogle Scholar
- Meyer, H.E., Hoffmann-Posorske, E., Donella-Deana, A., Korte, H., 1991 Sequence analysis of phosphotyrosine-containing peptides in Methods in Enzymology (Hunter, T., and Sefton, B.M., Eds.), Vol. 201, Academic Press, Orlando, FL.Google Scholar
- Wettenhall, R.E.H., Aebersold, R., Hood, L.E., Kent, S.B.H., 1991 Solid-phase sequencing of 32P labeled phosphopeptides at picomole and subpicomole levels in Methods in Enzymology (Hunter, T., and Sefton, B.M., Eds.), Vol. 201, Academic Press, Orlando, FL.Google Scholar