Edman reactions

THE EDMAN REACTION ENABLES PEPTIDES PROTEINS TO BE SEQUENCED... 

Figure 4-6. The Edman reaction. Phenylisothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenyithiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.

The Edman reaction enabled amino acid sequence analysis to be automated. Mass spectrometry provides a sensitive and versatile tool for determining primary strucmre and for the identification of post-translational modifications. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

Derivatization also allows specific residues to be detected and located by comparing the spectra before and after derivatization. The derivatizations most often used for this purpose are the formation of ethyl esters from Asp, Glu and C-terminal carboxylic acids acetylation of the N-terminal amino group and Lys and the Edman reaction for the N-terminal amino acid [60],... 

The MW determination by means of MALDI-TOF is so exact that the mass difference between a peptide with n amino acids and the same peptide minus one amino acid allows the identification of the missing amino acid. Even Asp (MW 115.1) and Asn (MW 114.1) can be determined this way. High-end devices even detect whether the peptide is lacking Lys (MW 146.190) or Gin (MW 146.146). However, the peptides should not be too large (maximum 30 amino acid residues). Following Chait et al. (1993), ladder sequencing is based on the accuracy of MALDI-TOF and a modified Edman reaction (Figure 7.10). 

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