Peptide Sequencing and Identification

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Peptides much longer than about 50 residues cannot be reliably sequenced by the Edman method because not quite all peptides in the reaction mixture release the amino acid derivative at each step. 

If the efficiency of release of each round were 98%, the proportion of “correct” amino acid released after 60 rounds would be only 0.3 (0.98) a hopelessly impure mix.  This obstacle can be circumvented by specifically cleaving a protein into peptides not much longer than 50 residues. 

In essence, the strategy is to divide and conquer.

Specific cleavage can be achieved by chemical or enzymatic methods. 

For example, Bernhard Witkop and Erhard Gross discovered that cyanogens bromide (CNBr) splits polypeptide chains only on the carboxyl side of methionine residues.

  A protein that has 10 methionines will usually yield 11 peptides on cleavage with CNBr.  Highly specific cleavage is also obtained with trypsin, a proteolytic enzyme from pancreatic juice, Trypsin cleaves polypeptide chains on the carboxyl side of arginine and lysine residues. 

 

A protein that contains 9 lysines and 7 arginines will usually yield 17 peptides on digestion with trypsin.  Each of these tryptic peptides, except for the carboxyl-terminal peptide of the protein, will end with either arginine or lysine.

The peptides obtained by specific chemical or enzymatic cleavage can then be separated by chromatography.  The sequence of each purified peptide is then determined by the Edman method.  At this point, the amino acid sequences of segments of the protein are known, but the order of these segments is not yet defined. 

The necessary additional information is obtasined from overlap peptides.  A second enzyme is used to split the polypeptide chain at other linkages.  For example, chymotrypsin cleaves preferentially on the carboxyl side of aromatic and some other bulky nonpolar residues.  Because these chymotryptic peptides overlap two or more tryptic peptides, they can be used to establish the order of the peptides.  The entire amino acid sequence of the polypeptide chain is then known.

These methods apply to a protein consisting of a single polypeptide chain devoid of disulfide bonds.  Additional steps are necessary if a protein has disulfide bonds or more than one chain.  For a protein made up of two or more polypeptide chains held together by noncovalent bonds, denaturing agents, such as urea or guanidine hydrochloride, are used to dissociate the chains.  The dissociated chains must be separated before sequence determination can begin.  Polypeptide chains linked by disulfide bonds are first separated by reduction with thiols such as beta-mercaptoethanol or diothiothreitol.  To prevent the cysteine residues from recombining, they are then alkylated with iodoacetate to form stable S-carboxymethyl derivatives.