The alkylated thiohydantoin method for C-terminal sequence analysis.

Abstract

The alkylated-thiohydantoin method for C-terminal sequencing makes a significant improvement to the thiohydantoin method first described by Schlack and Kumpf. Prior to cleavage from the protein, the C-terminal thiohydantoin is alkylated, making it a better leaving group than the unmodified thiohydantoin. The C-terminal alkylated-thiohydantoin can be cleaved from the protein under conditions that simultaneously form the next thiohydantoin. Combining cleavage and thiohydantoin formation in one step eliminates the need for activating the C-terminal carboxyl group before every sequencing cycle and prevents detection of C-termini formed by random cleavage of peptide bonds in the protein during the sequencing chemistry. The alkylated-thiohydantoin method includes the presequencing modification of cysteine and lysine and the automated modification of aspartic and glutamic acids, serine and threonine. Modifying the reactive side-chain groups improves the ability to sequence through and detect these amino acids. The alkylated-thiohydantoin method can sequence through and detect 19 of the 20 genetically coded amino acids. Sequencing stops at proline residues.