Benefits of using Edman degradation for amino acid sequencing
(1) Guaranteed N-terminal sequence of proteins
(2) No sample pre-treatment required
(3) Differentiation of isobaric amino acids (leucine and isoleucine)
(4) Identification possible even from unknown proteins not registered in databases
Protein Sequencer Analysis Example
Identification of more than 20 residues from 10 pmol of protein using differential chromatograms
Fig. 1 Example of Analyzing 10 pmol of BSA (Bovine Serum Albumin) by Isocratic system.
Only Cycle 1 is a raw chromatogram. The other two are differential chromatograms.
The number of cycles indicates the number of residues from the N-terminal. Cycle 1 shows a chromatogram for PTH-amino acid at the N-terminal, where D (aspartic acid) can be identified. DMPTU and DPTU are reaction byproducts of Edman degradation. DTT (dithiothreitol) is a reducing agent included in the reaction reagent. Except for Cycle 1, the chromatograms are differential chromatograms that were generated by subtracting the chromatogram from the preceding cycle from the chromatogram obtained in the current cycle. Differential chromatograms make it easier to identify amino acid sequences because they cancel out reaction byproducts and background peaks and clearly show selectively amplified PTH-amino acid peaks. These results show that the 15th and 22nd residues are G (glycine) and L (leucine), respectively.
Clearly identifies leucine and isoleucine in sequence
Fig. 2 Example of Analyzing 50 pmol Bovine Myoglobin by Isocratic system.
Cycles 11 and 21 are differential chromatograms.
The left chromatogram is an example of analyzing a standard PTH-amino acid mixture. Isoleucine and leucine are detected at different times. Both the 11th residue from the bovine myoglobin, which is L (leucine), and the 21st residue, which is I (isoleucine), are clearly identified.