The Complete Guide for The Principle and Steps of Protein Sequencing
Protein sequencing is the practical process of determining the amino acid sequence of all or part of a protein or peptide, which can be used to identify the protein or characterize its post-translational modifications. It mainly refers to the determination of the primary structure of the protein. The primary structure of the protein comprises the number of polypeptide chains that make up the protein.The main strategy for protein sequencing is to divide the polypeptide chain by chemical or enzymatic digestion and then determine the amino acid residue content and composition.
Protein Sequencing Steps
1. Cleavage of polypeptide chains. Protein molecules which is consisted of multiple polypeptide chains must be resolved first. Several polypeptide chains are linked together by noncovalent bonds, called oligomeric proteins, such as hemoglobin tetramer, enolase dimer; can be treated with 8 mol / L urea or 6 mol / L guanidine hydrochloride Separate polypeptide chains.
2. Determination of the number of polypeptide chains in the protein molecule. The number of polypeptide chains can be determined by measuring the relationship between the number of moles of terminal amino acid residues and the molecular weight of the protein.
3. Disulfide bond. Several polypeptide chains are cross-linked by disulfide bonds and can be treated with excess beta-mercaptoethanol in the presence of 8 mol / L urea or 6 mol / L guanidine hydrochloride to reduce the disulfide bond to mercapto. The resulting thiol group should be protected with an alkylating agent to prevent it from being reoxidized.
4. Dertermine each polypeptide chain of the amino acid composition and caculate the molecular ratio of the amino acid component.
5.Analyzing N-terminal and C-terminal of the polypeptide chain. Peptide chain end group amino acids are divided into two classes: amino-terminal and Carboxyl-terminal. In the peptide chain amino acid sequence analysis, the most important is the N-terminal amino acid analysis. N-terminal analysis (Sanger method; Edman method; DNS-Cl; enzymatic degradation), C-terminal analysis (hydrazinolysis; enzymatic degradation; lithium borohydride).
6. The polypeptide chain breaks into multiple peptides. The peptide sample can be broken into two or more sets of peptide fragments or peptides by two or more different fracture methods.
7.Determining the amino acid sequence of each peptide.
8. Determining the order of peptides in the polypeptide chain. The amino acid sequence of the entire polypeptide chain is interspersed with the overlapping of the amino acid sequences of two or more sets of peptides.
9. Determine the position of the disulfide bond in the original polypeptide chain. Generally, pepsin is used to treat the peptide chain without disconnecting the disulfide bond. And the peptide is separated by two-dimensional electrophoresis which can analyze and sequence the the peptide group that may contain the disulfide bond after treatment with formic acid. Methods were analyzed for peptide comparisons to determine the position of disulfide bonds.
N-terminal sequencing service
Almost all of the protein synthesis starts at the N-terminus, and the N-terminal sequence of proteins has a great influence on the biological function of the protein as a whole. For example, N-terminal sequence affects the half-life of proteins, meanwhile, it is associated with protein subcellular organelles. These are closely related to the function and stability of proteins. N-terminal sequencing of proteins is helpful to analyze the high-level structure of proteins and reveal the biological functions of proteins.
At present, the N-terminal sequencing of proteins is mainly classified into two major categories: non-mass spectrometry and mass spectrometry. Traditional non-mass spectrometry includes classical Edman degradation method that takes advantage of transcription-RT-PCR to get cDNA of corresponding protein, and then protein sequence will be obtained by reverse counting. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. Each of which has its own strengths and constraints.
All the above is about the details of protein sequencing which can be applied for protein identification, also will be helpful for studying the biological function of the peptide/protein