The Logic of Sequence Determination

A specific example will illustrate the reasoning that is used to fully determine the amino acid sequence in a particular peptide with 30 amino acid units. 

First we hydrolyze the peptide completely, subject it to amino acid analysis, and find that it has the formula 

Using the Sanger method, we find that the N-terminal amino acid is Phe. 

Since the chain is rather long, we decide to simplify the problem by digesting the peptide with chymotrypsin. (We select chymotrypsin because the peptide contains three Phes and two Tyr s and will undoubtedly be cleaved by that reagent.) When we carry out this cleavage, we get three fragment peptides. In addition, we get two equivalents of Phe and one of Tyr. After separation, we subject the three fragment peptides to Edman degradation and obtain their structures. 

We still cannot write a unique structure for the intact peptide, but we can say that the G-terminal amino acid must be Ala and that the last four amino acids must be in the sequence shown for fragment G. We deduce this because we know that Ala is not cleaved at its carboxyl end by chymotrypsin, yet it appears at the G-terminal end of one of the fragments. (Note that the G-terminal amino acids in fragments A and B are Phe and Tyr, both cleaved at the carboxyl ends by chymotrypsin.) That the G-terminal amino acid is Ala can be confirmed using carboxypeptidase. We can number the amino acids in fragment G as 27 through 30 in the chain. 

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