AUTOMATED PROTEOLYTIC MAPPING OF PROTEINS

A new, rapid, automated method for peptide mapping has been developed that requires less than two hours to complete. This method first (i) r educes the protein with dithiothreitol (DTT) or beta-mercaptoethanol a t 50 degrees C, (ii) then alkylates it with an alkylating agent select ed from iodoacetamide, iodoacetic acid, or vinylpyridine, (iii) digest s the protein completely with immobilized, TPCK treated trypsin, and ( iv) finally analyzes the tryptic fragments by high resolution, reverse d-phase liquid chromatography (RPLC) in less than two hours. Reduction and alkylation are achieved in the autosampler of the instrument wher e the sample, reagents, and reaction protocol are specified by the ope rator in the system computer. Proteins with up to seven disulfide brid ges were quantitatively reduced and alkylated by the system. Immobiliz ed enzyme columns coupled in tandem with an RPLC column were shown to generate protein digests and reproducibly separate the fragments for m any cycles of analysis. Based on the fact that any one of several alky lating agents could be used in the mapping process, it was demonstrate d that a campaign of experiments could be executed automatically in a search for the optimum alkylating agent. The mapping technique was app lied to five different proteins.