Chemical method refers to the method of specifically cleaving and detecting the C-terminal amino acids of proteins or polypeptides after they react with chemical reagents. There are mainly isothiocyanic acid method and perfluoric anhydride method. (custom peptide synthesis ) Other chemical methods, such as hydrazinolysis and reduction, are not commonly used because they are not easy to sequence consecutively, and will not be introduced.

Principle, characteristics and application of (ISO) thiocyanic acid process

(isothiocyanic acid method, also known as schlack kumpf degradation, is similar to Edman's degradation principle. That is, chemical reagent ((isothiocyanic acid) reacts with the O / monocarboxyl group of protein or polypeptide to form protein or polypeptide hydantoin, and then cleaves the C-terminal residue with cleavage reagent to form amino acid hydantoin. Then, the free amino acid hydantoin is separated and analyzed by HPLC system. Because the retention time of hydantoin of different amino acids is different on HPLC( exenatide impurities), various amino acids can be distinguished. The degradation of (isocyanate) by thiocyanic acid generally includes four steps J, namely carboxyl activation, coupling reaction, cyclization reaction and pyrolysis reaction. Although the results of each step are basically the same, each reaction has different ways of realization. There has been a special review. It is worth mentioning that the second step in the 4-step reaction -- coupling reaction. The reaction efficiency has always been a bottleneck of the (isothiocyanic acid) process.

The biggest difference between (isothiocyanic acid method and Edman degradation is also the different mechanism of coupling reaction. The coupling reaction of Edman degradation is to use the nucleophilicity of the N-terminal amino group of the protein to react with the electrophilicity of the isothiocyano carbon atom in the phenyl isothiocyanate (PITC) molecule to generate thiourea derivatives; The (isothiocyanate) law is to use the electrophilicity of the carboxyl carbon atom at the C-terminal of the protein to react with the thiocyanate ion or the nucleophilicity of the isothiocyanate nitrogen atom to generate isothiocyanate derivatives. Due to the great difference in chemical activity between the amino nitrogen atom and the carboxyl carbon atom, the coupling yield of (isothiocyanic acid method is hardly comparable to that of Edman degradation. With acetic anhydride as the activation reagent, tetrabutylammonium thiocyanate as the coupling reagent, and bromomethylnaphthalene as the alkylation reagent, the C-terminal of protein or polypeptide is successively converted and cleaved into ath amino acid, and the detection is carried out at 254nm. They have carried out C-terminal analysis on more than 20 recombinant or natural proteins or polypeptides, with molecular weights ranging from 2 to 60 kD. They have obtained C-terminal sequence information of different lengths. Generally, they can determine 3 to 5 residues at the level of 12 nmol, and up to 10 residues for myoglobin. Acetic anhydride is used as the activation reagent, the difference is that triphenylmethane isothiocyanate (tpg-itc) is used as the derivatization reagent, and NaOH is used as the cracking reagent. They used this method to obtain eight amino acid residues of a sixteen peptide at the nmol level

In short, this method has the advantage of automatic and direct determination of the C-terminus of proteins or polypeptides. However, due to the limitation of coupling reagent and cracking reagent, the final yield and repetitive yield of this method are low. It is reported that the number of residues obtained by this method is not large, and the vast majority are less than 10 residues. At the same time, the method has some disadvantages, such as low sensitivity (20 ~ 100pmo1), and difficulty in determining special amino acids, such as proline