Practical Protein Chemistry - A. Darbre 1989

Determination of the amino acid sequence of peptides by the Edman method with identification of dansyl amino acids
Edman condensation and cleavage
Edman degradation. Methodology

1. Phenylisothiocyanate, pyridine, and trifluoroacetic acid are stored in tightly sealed containers under nitrogen (nitrogen purging must be repeated before and after using these Reagents).

2. Dry 20–50 nmol of peptide completely (the sample must be salt-free).

3. Add 100 µl of 50% pyridine and flush with nitrogen for 1 min.

4. Add 50 µl of 10% PITC in pyridine and flush with nitrogen again for 1 min.

5. Seal the ampoule and incubate in a heating block at 50 °C for 30 min.

6. Place the vacuum desiccator containing phosphorus pentoxide and sodium hydroxide in an incubator at 60 °C.

7. Remove the desiccator from the incubator and connect it to a vacuum line to dry the Peptides.

8. After 10 min, check whether a pinkish solid residue (consisting of the PTC-peptide and diphenylthiourea mixture) has formed. If the residue remains oily, add 50 µl of 95% ethanol and repeat the drying Procedure.

9. Dissolve the precipitate in 50 µl of anhydrous trifluoroacetic acid, close the tube, and incubate for 15 min at 50 °C. If an N-terminal glutamine residue is expected to form upon Cleavage, reduce the incubation time to 5 min (this minimizes the risk of glutamine cyclization into pyrrolidonecarboxylic acid).

10. Dry the peptide over sodium hydroxide, add 50 µl of ethanol if necessary, and evaporate to dryness again.

11. Add 100 µl of deionized Water. Thoroughly mix with 1 ml of butyl acetate using a vortex mixer and separate the phases in a benchtop centrifuge for 5 min. Remove the organic phase, taking great care not to disturb any precipitate of insoluble peptide that may form at the phase boundary. Repeat the butyl acetate wash twice.

12. Evaporate the aqueous phase to dryness and dissolve it in 1% triethylamine. Take an aliquot for Dansylation, dry down the remainder, and proceed to the next cycle of Edman Degradation. If the procedure needs to be interrupted, this should always be done after the cleavage step.

Note. To speed up sample drying, a vacuum centrifuge (Speed Vac Concentrator by Savant Instruments) may be used.