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Protein Sequencing
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N-terminal sequencing: Edman’s approach
This procedure involves the sequential (i) chemical modification of the N-terminal residue, through its amino group with phenylisothiocyanate (PITC), (ii) removal of NH2-derivatized amino-acids from the N-terminal to the C-terminal of a peptide chain, and (iii) reverse phase HPLC analysis with UV detection at 270 nm.
Comparison of the successive elution profile to that of a standard set of adducted amino-acids allows for sequence calls (Fig.1).
This procedure involves the sequential (i) chemical modification of the N-terminal residue, through its amino group with phenylisothiocyanate (PITC), (ii) removal of NH2-derivatized amino-acids from the N-terminal to the C-terminal of a peptide chain, and (iii) reverse phase HPLC analysis with UV detection at 270 nm.
Comparison of the successive elution profile to that of a standard set of adducted amino-acids allows for sequence calls (Fig.1).
- Fig 1. HPLC elution profile of a standard mixture of 19 adducted amino-acids providing standard retention times. Several by-products, indicated by an X, are formed during the Edman degradation process.
- Click on figure to enlarge
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Internal sequencing by chemical cleavage: please submit your project details for evaluation.