Darryl J. C. Pappin

Synthesis, Evaluation and Application of a Panel of Novel Reagents for Stepwise Degradation of Polypeptides

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.