Sequencing Proteins:

General strategy:

1. Prepare a pure sample of the protein of interest.

2. Cleave the protein into a number of defined peptides. The sequencing of all biological polymers requires that they be broken down into smaller segments, since one can't usually sequence the whole macromolecule in one step.

3. Purify these peptides (separate by column chromatography).

4. Sequence each peptide using the Edman degradation.

5. Repeat steps 2, 3, and 4 (above) with peptides from the same protein obtained by using a cleavage agent with a different specificity (produces a different set of peptides).

6. Determine order of peptides in the native protein by using the technique of peptide overlap:

N__________ ______________ ______C

N_____ ___________ ______________C

7. Write out the complete sequence.

Peptide cleavage reagents:

1. Cyanogen bromide: one of the most specific and useful reagents for the biochemical cleavage of peptide bonds. Cleaves on the carboxyl side of methionine residues.

2. Trypsin: cleaves on the carboxyl side of Lys and Arg

3. Pepsin: cleaves on the carboxyl side of Phe, Leu, Trp, or Tyr

4. Chymotrypsin: cleaves on the carboxyl side of Phe, Trp, Tyr, Ile, Val, Leu

5. Thrombin: cleaves on the carboxyl side of Arg

Ion-exchange chromatography is employed to separate peptides, some of which may be very insoluble in water. This type of chromatography separates on the basis of charge.

Edman Degradation:

The amino acid sequence of a protein or peptide can be determined using the organic chemical reagent phenylisothiocyanate.

1. can't sequence entire macromolecules.
2. cleavage into small segments requires reagents that act cleavage at specific sites.
3. must be able to separate the fragments (chromatography for proteins; cloning for DNA)
4. must have chemistries that permit identification of a single AA or base (or a difference of one at a time).