Sanger’s labor-intensive approach rendered it prohibitively difficult to apply to any but the smallest polypeptides. Pehr Edman discovered a reagent, phenyl isothiocyanate (Edman reagent), that not only could selectively label the amino-terminal residue of a peptide as its phenylthiohydantoin (PTH) derivative but, in contrast to Sanger reagent, permitted the PTH derivative to be removed under mild conditions (Figure 1). The new amino terminal residue could then be treated with Edman reagent and the process repeated to permit each successive residue in a peptide to be derivatized.
Fig1. The Edman reaction.Phenyl isothiocyanate derivatizes the amino-terminal residue of a peptide as a phenyl thiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenylthiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.
While, in theory, one could determine the entire sequence of a polypeptide using Edman reagent, the heterogeneous chemical properties of the amino acids meant that every step in the procedure represented a compromise between efficiency for any particular amino acid or set of amino acids and the flexibility needed to accommodate all 20. Consequently, each step in the process operates at less than 100% efficiency, which leads to the accumulation of polypeptide fragments with varying N-termini that eventually renders it impossible to distinguish the correct PTH amino acid for that position in the peptide from the out-of-phase contaminants. As a result, the read length for Edman sequencing varies from 5 to 30 amino acid residues depending on the quantity and purity of the peptide, hardly enough to determine the sequence of a typical protein.
To determine the complete sequence of a polypeptide several hundred residues in length, a protein must be cleaved into smaller peptides. These were then purified and analyzed by Edman sequencing. This yielded multiple segments of sequence whose location within the protein, with the exception of the amino terminus, was unknown. In order to assemble these short peptide sequences into the complete sequence of the intact polypeptide, it was necessary to generate and analyze additional peptides in search of some whose sequences overlapped with one another, thereby allowing larger segments of sequence to be pieced together. While the development of automated Edman sequencers and sophisticated HPLC systems for purifying peptides often rendered initial acquisition of a fragmentary, or partial, sequence relatively quick, finding enough “overlap” peptides to reconstruct the complete sequence of a typical protein via the Edman technique generally required large quantities of purified protein and, more importantly, several months or years of additional work. Consequently, the determination of a complete amino acid sequence via the Edman method generally was restricted to highly abundant, readily purified proteins.
