title: 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.
authors.affiliation: PE Biosystems, Foster City, California 94404, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Dupont
authors.forename: M
authors.initials: M
authors.lastname: Bozzini
authors.forename: V L
authors.initials: VL
authors.lastname: Boyd
mesh_terms: D000478:Alkylation; D000596:Amino Acids / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013867:Thiohydantoins / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D011506:Proteins; D013867:Thiohydantoins