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[27] Automatic solid-phase Edman degradation

1972; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(72)25030-3

1557-7988

Richard A. Laursen,

Machine Learning in Bioinformatics

This chapter discusses that in Edman's automatic protein sequenator, proteins are immobilized as a thin film in a spinning cup, and separations are accomplished by a selective extraction procedure. The solid-phase modification differs in the sense that peptides are bound covalently to an insoluble resin that can be washed free of excess reagents. The first step of the solid-phase method is to attach peptides by their carboxyl termini to the resin. Peptide amino groups are blocked using t-butyloxycarbonyl azide, and the carboxyl groups are activated using carbonyldiimidazole. The resulting peptide imidazolides are then coupled to an aminopolystyrene resin. Deblocking of the amino groups is accomplished by treating the resin with trifluoroaeetic acid. Degradation of the peptide is carried out in the usual way by treatment with phenyl isothiocyanate, followed by trifluoroacetic acid. The thiazolinones liberated in each stage are isomerized to phenylthiohydantoins and are identified using an isotope dilution procedure. The solid-phase peptide sequencer is designed with the aim of providing a simple, rapid, and relatively inexpensive means for sequencing peptides of the size normally encountered in enzymatic digests of proteins.