On ‘A method for the determination of amino acid sequence in peptides’ by P. Edman

Protein detection can be done by Western blotting, but the most accurate method to identify a protein is to utilize a protein sequencing method. The two main methods for protein sequencing are Edman degradation and mass spectrometry. Accurately identifying proteins in complex mixtures have significantly contributed to our understanding of cellular function. Edman degradation, the first method to determine the amino acid sequence of a peptide, was published in 1949 in the Archives of Biochemistry [1]. In 2022, Edman degradation is alive and well because it complements mass spectrometry sequencing techniques and has an advantage over mass spectrometry in sequencing peptides from animals for which no sequence database is available.

In 1945, the Sanger group published a method to determine the N-terminal residue of a peptide using fluorodinitrobenzene (FDNB) [2]. The limitation of this method was that it only determined the free N-terminal amino acid of a protein or peptide. A few years later, in 1949, Pehr Victor Edman published the first method to sequence a protein. Dr. Edman was 33 years old when he published this groundbreaking paper that was only 300 words long (not counting author’s name and reference) [1]. In 1950, Edman published a more detailed report about the Edman reaction [3]. This method, which was later called the Edman degradation method, involved a cyclic chemical reaction that allowed the N-terminal residues of a protein to be determined. The key to Edman’s method was the reagent phenylisothiocyanate (PITC), later called Edman’s Reagent. At alkaline pH PITC interacts readily with N-terminal amino acids of peptides resulting in phenylthiocarbamyl amino acid derivatives. Removal and identification of the labeled N-terminal amino acid without disrupting the peptide bonds of other residues in the peptide being sequenced allows the N-terminal sequence to be accurately determined.

During this same time period Fredrick Sanger continued to improve his method for sequencing proteins and started using partial hydrolysis, proteolytic enzymes and different types of chromatography. In 1953, Sanger published the sequence of bovine insulin and was awarded the Nobel Prize in 1958 for his work on sequencing [4,5]. The Edman reaction was also being improved, and like many great methods, the next step in the Edman degradation method was automation. Edman and Beggs perfected an automated version of the Edman degradation method in 1967 [6]. At first, only a few individual laboratories had the capability to do automated Edman degradation, but by 1973 about 100 laboratories worldwide had this capability [7]. These automated protein sequences were reported to be able to sequence 60–150 amino acids from the N-terminal [7]. In 1982, the first commercially available Edman degradation machine (from Applied Biosystems) was available. This corresponded with the increase in the number of publications using this technique. However, once mass spectrometry methods were developed that allowed protein sequencing, the popularity of Edman degradation sequencing decreased. A timeline of the major advances involved in protein sequencing is shown in Fig. 1.

Fig. 1.

Timeline of major events in the development of protein sequencing.

As life would have it, Edman degradation is back in the limelight. Swaminathan et al. developed a novel automated, high sensitivity, ultrarapid proteomic technique that utilizes aspects from single molecule DNA sequencing, Edman reaction, and mass spectrometric database searching [8]. Fluorescently labeled peptides were immobilized onto a glass coverslip at the C-terminus, and N-terminal amino acids were removed by sequential cycles of Edman degradation. As amino acids were removed the loss of fluorescently labeled amino acids were monitored. The strength of this technique is its sensitivity and ability to potentially sequence thousands of peptides at the same time. In 2022, several companies, including Proteome Factory, Berlin, and Alphalyse, USA currently offer Edman degradation sequencing commercially, and many research core facilities offer Edman degradation sequencing as a service. As such, the robustness of Edman’s chemistry has allowed it to be still valuable to scientists after over 70 years since it was introduced.