N-Terminal Sequencing

What is N-Terminal Sequencing?

N-Terminal Sequencing (also known as Edman Degradation) is a method developed by Pehr Edman, a Swedish biochemist, for obtaining the amino acid sequence of peptides. Using this method, the n-terminal amino acid residue is labeled and removed from the subject peptides that constitute a pure sample. These free amino acids are then subjected to C18 reverse phase chromatography coupled to a detector. Eluting residues are identified by their characteristic retention time. The process is then repeated for each new n-terminal residue until the desidered sequence has been elucidated.

The Proteomics and Metabolomics Facility at Colorado State University houses an Applied Biosystems 491 Procise protein sequencer which automates the Edman Degradation process.

Details of Edman Degradation

Step 1 and 2: The N-terminus of the protein or peptide is reacted with phenylisothiocyanate (PITC) to form a phenylthiocarbamyl derivative (PTC-protein).

Step 3: Trifluoroscetic acid is used to cleave off the first amino acid on the N-terminus as its anilinothialinone derivative (ATZ-amino acid). This process leaves a new free amino terminus for the next degradation cycle.

Step 4: The ATZ amino acid is then removed by extraction with N-butyl chloride and converted to a phenylthiohydantoin derivative (PTH-amino acid) with 25% TFA/water

Step 5: The PTH-amino acid is transferred to a reverse-phase C-18 column for detection at 270nm. To determine the amino acid present in each cycle, the chromatograms are compared to the chromatogram of the amino acid standards.

Image animation from http://www.protein.iastate.edu/nsequence494.html

Sample Submission

The minimum recommended sample amount for n-terminal sequencing is 10pmol, however 100pmol is optimal for best results.

The best sample preparation is gel separation, electroblotting onto PVDF and staining with Coomassie Blue. Samles may also be submitted in liquid form and we will purify them using the ABI ProSorb system (additional sample preparation fee of $10 will apply).

Please note that many native proteins are N-terminally modified in which case Edman degradation will yield no sequence information. Full analysis costs will still apply to cover instrument use and reagents.

Please print and submit a sample submission form with your sample.