Modular Berlin Microsequencer for the Sequential Degradation of Proteins and Peptides from the Amino- and Carboxyl-Terminal End

Summary

Based on the idea of designing automates in a modular construction Berlin sequencers for stepwise degradation of polypeptides from their N-terminal amino acid have been developed. With these parts a new sequencer which allows to degrade peptides and proteins from their C-terminal end has been assembled. Programs that allow to sequence polypeptide chains either by the Edman chemistry (employing phenylisothiocyanate) from the N-terminal end, or alternatively, by a Schlack-Kumpf-type chemistry using isothiocyanate reagents for sequencing from the carboxyl-terminal amino acid were applied. Degradations from the C-terminal end were performed in three different type of machines, a laboratory constructed liquid-phase cup sequencer, a solid-phase machine equipped with the commercial available Knauer-sequencer modules in combination with a new reactor for quick heating and cooling, and another type equipped with a flow-through block reactor that allows to degrade proteins without polybrene on PVDF, glass fibers or derivatized membranes. In all these C-terminal sequencers both, an isocratic and gradient HPLC were tested for the on-line in-situ identification of the released thiohydantoin-amino acid derivatives.