Edman degradation is a chemical method used for the sequencing of amino acids in a
peptide. This method is developed by Pehr Edman in 1960. In this methods, N-terminal or
amino terminal is labeled or cleaved from the peptide and number of residues are identified.
Process
• Suppose we have a sequence MYKMRYY.
• We will label amino terminal with isothiocynate (component of certain plants and
vegetables).
• Then we will do mild hydrolysis, on hydrolysis only labeled amino acids will be separated.
• Through HPLC, High Pressure Liquid Chromatography, we will identify this labeled amino
acid.
• Then again label the N-terminus of remaining polypeptide chain.
• All the above steps will be performed for all amino acids.
Disadvantage of Edman Degradation
• It is very exhaustive method.
• Start with N-terminus, if protein not linear and N-terminus inward or if N-terminus is
modified then reagent will not bind, we will not proceed.
• In 24 hours only 10 residues identified.
• More protein sample is required.
• Upto 50-60 residues this process is efficient and accurate, above this, its efficiency
reduced.
Advantage of Edman Degradation
• 100 % surety of finding protein sequence.
To solve or increase efficiency
• To solve or increase efficiency we will fragmentize whole polypeptide.
• Suppose we have 400 peptides, fragmentize this 400 peptides, using different enzyme like
trypsin. It cuts at basic amino acid (Arginine/ lysine) if next amino acid is not proline.
• Arginine/lysine amino acid exists commonly.
• In 500 amino acids protein, there is chance of 20-25 sites to fragmentize.
• So all fragments will be of different and unknown size.
Problem: We do not know about the arrangement or sequence of these fragments.
Two ways to solve problem
• Uses different reagents to cut the peptides/multiple proteases.
• Fragment assembly or assemble fragment.
Assemble fragments
One sequence is digested with trypsin. After digestion these fragments generates.
1. Lue-Asp-Glu-Tyr-Gly-Val-Ile-Lys.
2. Ala-Val-Ile-Leu-Ser-Glu-Ile-Leu.
3. His-Thr-Val-Glu-Val-Arg.
The same sequence will be digested now with Glu-C. The following fragments will be
generated.
1.
2.
3.
4.
Leu-Asp-Glu.
Tyr-Gly-Val-Ile-Lys-Ala-Val-Ile-Leu-Ser-Glu.
Ile-Lys-His-Thr-Val-Glu.
Val-Arg.
By overlapping these fragments we will find out which fragment will be next for example
compare fragment of trypsin digestion and fragment of Glu-C digestion. We will know first
three amino acids.
Overlapping sequences (fragments) can be sequenced following digestion of the protein with
a reagent with different specificity to trypsin. It is important to choose a reagent with a very
long and specific recognition site.
The solved amino acid sequences of peptides can be used to design degenerate PCR primers
which can be used to isolate a corresponding genomic or cDNA sequence. This can then be
translated to predict the full length protein sequence.