The C-terminal sequence is an important structural and functional site of proteins and polypeptides, and even plays a decisive role in the biological functions of proteins. In addition, C-terminal splicing is also one of the important post-translational modifications of proteins. With the deepening of proteomic research, protein C-terminal sequencing will play an increasingly important role in the study of its function. Some new protein C-terminal sequencing methods have been established, which improve the sensitivity and repeatability and can be applied at the proteome level.

The primary structure of protein determines the higher structure, and the higher structure determines the function. Therefore, the primary structure of protein must be determined to study its function. To determine the primary structure of a protein, it is generally necessary to determine its molecular weight, isoelectric point, N-terminal sequence and C-terminal sequence. At present, the methods of protein molecular weight determination, isoelectric point determination and N-terminal sequence determination are mature, with high sensitivity and accuracy. However, the technology of C-terminal sequencing is still immature, which is the bottleneck of protein primary structure research and needs further development.

With the development of proteomics, the post-translational modification of proteins has become the focus of scientists' attention. Like phosphorylation and glycosylation, N-terminal and C-terminal cleavage of proteins are also important post-translational modifications of proteins, which play an important role in protein function. Therefore, studying the N-terminal and C-terminal sequences of proteins is beneficial to reveal the biological functions of proteins. At present, the protein N-terminal sequencing method is mainly Edman degradation method. This technology is very mature and automatic, and is widely used in proteomic research. However, proteomics based studies have rarely determined the C-terminal sequence of proteins, mainly because there is no mature one that can be widely used

Protein C-terminal sequencing method applied to proteomic research. Two dimensional electrophoresis combined with mass spectrometry is the most widely used method in proteomic research. On the two-dimensional electrophoresis gel map, multiple protein spots are often identified as the same protein, which is caused by different post-translational modifications or cleavages, of which C-terminal cleavage is an important form of cleavage. Therefore, it is of great significance to establish a C-terminal sequencing method based on gel separation to elucidate the primary structure and biological function of proteins and promote the development of proteomics.

Protein C-terminal sequencing methods mainly include carboxypeptidase method, chemical method and tandem mass spectrometry. Each method has certain limitations. With the continuous development of mass spectrometry, carboxypeptidase and tandem mass spectrometry are more and more widely used

Carboxypeptidase method

Principle of carboxypeptidase method

Carboxypeptidase is an exoenzyme that releases amino acid residues one by one from the C-terminal of protein. The principle of carboxypeptidase method is to judge the C-terminal sequence of protein by detecting the sequence of amino acids released by carboxypeptidase one by one. Among them, there are two methods to detect the released amino acids: (1) chromatography: directly detect the released amino acids; (2) Mass spectrometry: infer the type of amino acid released by measuring the difference between the original molecular weight of protein or polypeptide and the molecular weight after releasing an amino acid, and so on. Carboxypeptidase is a widely used method for C-terminal sequencing of proteins and polypeptides in recent years. There are four kinds of Carboxypeptidases, namely, carboxypeptidase A (CPA), carboxypeptidase B (CPB), carboxypeptidase y (CPY) and carboxypeptidase P (CPP). CPY and CPP are commonly used

Characteristics and application of carboxypeptidase digestion chromatography

After the protein is digested by carboxypeptidase, the released amino acids are detected by high-pressure liquid chromatography at different time points, and the C-terminal sequence of the protein can be deduced from the sequence of the released amino acids. This method is often used in early work N-Fmoc-7-methyl-L-tryptophan. However, this method may be interfered by impurities contained in enzymes or proteins or polypeptides or amino acids cut by enzymes. A bigger obstacle is that different amino acids have different release rates, so to obtain accurate C-terminal sequences, many time points must be selected for determination. Because this method is time-consuming and laborious, it is rarely used now.

Characteristics, applications and progress of carboxypeptidase digestion mass spectrometry

Mass spectrometry has become a very important tool for studying proteins. Similarly, the development of mass spectrometry technology also provides favorable conditions for the C-terminal sequence analysis of proteins. Matrix assisted laser desorption /CBZ-OSu  ionization time of flight mass spectrometer (malditof-ms) has become the first choice tool for protein identification because of its high sensitivity, high resolution, high accuracy and simple operation. In C-terminal sequencing, MALDI-TOF-MS is also used most in combination with carboxypeptidase digestion