Solid-phase protein and peptide sequencing using either 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate or phenylisothiocyanate.

H S Lu, R W Gracy

Index: Arch. Biochem. Biophys. 235(1) , 48-62, (1984)

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Abstract

Automated solid-phase sequencing using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate (DABITC) double coupling or regular phenylisothiocyanate (PITC) degradation procedures have been investigated. Employing sensitive high-performance liquid chromatography for the identification of amino acid thiohydantoin derivatives (PTH and DABTH), both methods were capable of sequencing immobilized peptides or proteins at the subnanomole levels. In the sequencing program using DABITC, alternate methanol and dichloroethane washes and automated conversion using methanolic HCl containing dithiothreitol were introduced to obtain clean thiazolinones and to ensure high recovery yields of the thiohydantoins. Using regular PITC degradation with a 59-min program, the background peaks of the side products could be reduced to enhance HPLC identification. Peptides or proteins attached to the glass beads or resins via the carboxyl terminii or epsilon-amino groups of lysyl residues could be readily sequenced up to 30 identifiable degradation cycles, where the sequencing is generally terminated due to the increased background components.