The chemistry of protein sequence analysis

N-terminal sequence analysis by Edman chemistry continues to play an important role in the structural analysis of proteins and peptides. Improvements in the sensitivity of the method have been achieved mainly at the level of increasing the sensitivity of the on-line analysis of PTH amino acids by RP-HPLC (reverse phase high performance chromatography). Using microbore columns (0.8-1.0 mm), it is possible to run standards at the 0.5-1.0 pmol level and to sequence samples in the 1-5 pmol range. Due to constraints in current chromatographic methods, it is unlikely that further improvements in sensitivity will be achieved by this approach alone. Although alternative Edman reagents, including fluorescent chemistries, have promised to increase the sensitivity of sequencing into the low femtomole range, none of the methods have progressed into routine usage. These reagents and chemistries are critically evaluated in this review, and the problems which have prevented their further development discussed. Instrumental constraints are also considered. It is concluded that the development of more sensitive methods requires further research into both the chemistry and the instrumentation, and that alternative separation and detection methods may also play a role.