1 You have purified a protein and are now trying to determin

1. You have purified a protein and are now trying to determine the sequence. Using the Edman Degradation method, you receive the results M,N,Y,I, G, R. When you digest a sample of your protein with Trypsin, you get the following fragments:

ASTFLMR GATWCDEK ICYLVSK MNYIGR NFHPPR MQL

When you digest a sample of your protein with Chymotrypsin you get a different set of fragments:

MNY LMRNF CDEKICY LVSKMQL IGRASTF HPPRGATW

a) How does the Edman Degradation method work?

b) What is the correct sequence of your protein?

c) What are alternate ways you could have determined the sequence of your protein?

Solution

Answer:

A) In Edman degradation method, the amino terminal residue is labeled by phenylisothiocyanate (PITC) and cleaved from the peptide , without disrupting the peptide bonds between other aminoacid residues.

B)

The sequnce of protein

MNYIGRLMRNFWCDEKICYLVSKMQL

C)

For protein sequencing ,the mass spectrometry is the best method.

Alternatively we can also use the sequence of the RNA or DNA as the back bone sequence of protein