Protein Sequencing
Edman Degradation Chemistry

There are 7 stages in N-terminal protein sequencing.

• COUPLING - Firstly phenylisothiocyanate (PITC, R1) reacts with the N-terminal amino acid residue under basic conditions (provided by n-methylpiperidine/methanol/water, R2) to form a phenylthiocarbamyl derivative (PTC-protein).
  
  
  
  
• WASHING - The PTC-protein is washed with ethyl acetate (S2) and n-heptane (S1) to remove excess PITC.
  
  
• CLEAVAGE - The PTC-protein is then treated with neat Trifluoroacetic acid (TFA, R3) to cleave the first amino acid forming an anilinothialinone derivative (ATZ-amino acid) leaving the new amino terminus of the protein for the next degradation cycle.
  
  
  
  
• EXTRACTION - Following cleavage the ATZ amino acid is extracted with N-butyl chloride (S3).
  
  
• CONVERSION - The ATZ amino acid is converted to a phenylthiohydantoin derivative (PTH-amino acid) with 25% TFA/water (R4) before separation by reverse phase hplc..
  
  
  
  
• RESOLUTION - The PTH-amino acid together with several by-products formed during the Edman degradation chemistry are injected onto a C-18. The retention time of the PTH amino acid is compared with the retention times of a standard mixture of 19 PTH-amino acids for identification. This standard chromatogram provides standard retention times of the amino acids for comparison with each Edman degradation cycle chromatogram.
  
  
• ANALYSIS - The HPLC chromatograms are collected using a computer data system for analysis. To determine the amino acid the chromatogram from the residue of interest is compared with the chromatogram from the previous residue by overlaying one on top of the other. The process is repeated sequentially to provide the N-terminal sequence of the protein/peptide.