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Edman Degradation - For Protein SequencingBY: Muniba Safdar | Category: Genetics | Submitted: 2010-08-10 06:15:56
Article Summary: "What is Edman Degradation method? It 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..."
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.
• 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.
One sequence is digested with trypsin. After digestion these fragments generates.
The same sequence will be digested now with Glu-C. The following fragments will be generated.
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.
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