High sensitivity automated sequence determination of polypeptides

Abstract

We report the development of a high sensitivity Edman method for use in the automated protein sequenator. With the use of a radioactive coupling reagent, [35S]phenylisothiocyanate, and minor modifications in the sequenator program, sequence analyses have been performed on nanomole quantities of protein. The radioactive phenylthiohydantoin derivatives produced in the degradation are identified at the 10 to 100 pmol level by two-dimensional thin layer chromatographic procedures with autoradiography and are quantitated by scintillation counting. This high sensitivity approach, which is about 100 times more sensitive than conventional automated Edman procedures, has allowed continuous amino acid assignments for 15 or more cycles on quantities of protein less than 5 nmol. It has been employed in the NH2-terminal sequence analysis of several proteins whose sequences were previously undetermined.