The alkylated thiohydantoin method for C-terminal sequence analysis.
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.
Dupont
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PE Biosystems, Foster City, California 94404, USA.
Bozzini
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M
Boyd
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VL
D000478:Alkylation; D000596:Amino Acids / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013867:Thiohydantoins / Q000737:chemistry*
D000596:Amino Acids; D011506:Proteins; D013867:Thiohydantoins