What is Amino Acid Analysis?

  • Amino acids are compounds in which hydrogen atoms on carboxylic acids are replaced by amino acids, and amino acids contain amino and carboxyl functional groups. Amino acids are the building blocks of protein for animal nutrition. It is an organic compound containing basic amino group and acid carboxyl group.

    Amino acids can play the following roles in human body through metabolism:

    ① compound the histone;

    ② Become acid, hormone, antibody, creatine and other ammonia containing substances;

    ③ Convert to carbohydrates and fats;

    ④ Oxidation to carbon dioxide, water and urea in order to produce energy.

    Amino acid analysis is a method used to determine the amino acid composition or content of proteins, peptides and other pharmaceutical preparations by physical and chemical means. Protein and peptide can be identified according to the composition analysis of amino acids. Amino acid analysis can be used to determine the content of proteins, peptides and amino acids. In addition the atypical amino acids that may exist in proteins and peptides can also be determined with it. Before amino acid analysis, proteins and peptides must be hydrolyzed into individual amino acids. Specific hydrolysis methods shall be specified under each variety. After protein and peptide hydrolysis, the process of amino acid analysis is the same as the analysis method of other free amino acids in pharmaceutical preparations.

    The following three methods are introduced for the analysis about amino acids.

    Host Cell Protein HCP Analysis

    The residual host cell protein (HCP) in biological products as an exogenous source may trigger the immune response of the body to varying degrees and eventually lead to allergic reaction or other adverse reactions. Therefore, an appropriate HCP detection method must be established to monitor the quality of biological products.

    We take Vero cells for example. The HCP of Vero cell refers to the protein components derived from host cells in a vaccine. Its main components include: host cell structural proteins and transformation proteins (somatotropin secreted by cells). The former is dangerous for inducing allergic reactions in the body, while the latter is potentially dangerous for inducing cell transformation.

    HCP is protein which is secreted into the culture medium by the residual cells that remains in the purification process, or structural protein produced from died broken cells. Before the advent of sensitive immunoassays, protein staining and immunoblotting were the test criteria and were considered adequate. Here are some common ways to analyze HCP: SDS-PAGE, HPLC, ELISA and so on. One common feature of the above methods is that the high concentration of biological products in the samples can easily mask the existence of small bands or protein spots. The detection method requires the identification of noise (HCP) and high overdose primary bands or differences of signal for protein spots. These methods are difficult to detect the existence of these trace HCP. At present, ELISA is still the most widely used HCP detection method, mainly because its technology is relatively simple and accurate, but also because it can provide quantitative and digital results, so as to set control range and establish technical specifications.

    De-novo protein sequence analysis

    De Novo Sequencing is two kinds of rapid sequence determination techniques currently used nowadays. The theory is to create separate groups of radio-labeled oligonucleotides, each of which has a fixed starting point but terminates randomly on a specific or multiple residue. Only by adding several groups of oligonucleotides to several adjacent swim lanes in the sequencing gel, the oligonucleotides of each group can be analyzed by electrophoresis under the condition of differentiating different DNA molecules with only one nucleotide difference in length.

    CDR region, is the core of its biological function, which is of great significance for the accurate and rapid analysis of its complete sequence. The first order sequence of the antibody can be determined by de Novo Antibody Sequencing, on which basis, the antibody affinity enhancement and humanization can be carried out to develop the antibody drugs. In the research and development of antibody imitation drugs, the accurate first-order structure information of the original drug is the basis and premise of the research and development of antibody imitation drugs.

    Shotgun Proteomics

    Shotgun proteomics strategy is a method of identifying proteins directly from complex protein mixtures without protein purification. Usually, proteins are digested into polypeptides and then sequenced by tandem mass spectrometry. The obtained polypeptide spectral data are used to search the database. SEQUEST software is used to analyze the first-order structure information of peptide fragments, so as to identify proteins. Shotgun proteomics, which is used to identify samples of mixed proteins with low copy, hydrophobic proteins and some polar proteins, has the advantage that two-dimensional gel electrophoresis (2- DE) technology cannot replace.

    In the course of vaccine research, the shotgun proteomics can be used to analyze the surface proteins of the source of infection, so as to find the potential antigen. It’s being accepted and applied by more and more scientists, so it will have more and more applications in the development of new drugs and the treatment of diseases in the near future

    In conclusion, amino acid analysis is of great importance in protein chemistry and biotechnology research, clinical diagnosis and cell physiology research, nutrition and health care, cultivation of good varieties of crops, food, pharmaceuticals and other fields.

    1.amino acid analysis: Amino acid analysis is a method used to determine the amino acid composition or content of proteins, peptides and other pharmaceutical preparations by physical and chemical means.

    Amino acid analysis can be used to determine the content of proteins, peptides and amino acids. In addition the atypical amino acids that may exist in proteins and peptides can also be determined with it. Before amino acid analysis, proteins and peptides must be hydrolyzed into individual amino acids. Specific hydrolysis methods shall be specified under each variety. After protein and peptide hydrolysis, the process of amino acid analysis is the same as the analysis method of other free amino acids in pharmaceutical preparations.

    1. host cell protein HCP analysis: An appropriate HCP detection method to monitor the quality of biological products.

    An appropriate HCP detection method to monitor the quality of biological products, the common ways usually are SDS-PAGE, HPLC, ELISA and so on. One common feature of the above methods is that the high concentration of biological products in the samples can easily mask the existence of small bands or protein spots. The detection method requires the identification of noise (HCP) and high overdose primary bands or differences of signal for protein spots. These methods are difficult to detect the existence of these trace HCP.

    1. de Novo Antibody Sequencing: A method to determine the first order sequence of the antibody.

    The first order sequence of the antibody can be determined by de Novo Antibody Sequencing, on which basis, the antibody affinity enhancement and humanization can be carried out to develop the antibody drugs. In the research and development of antibody imitation drugs, the accurate first-order structure information of the original drug is the basis and premise of the research and development of antibody imitation drugs.

    1. de-novo protein sequence analysis: It is two kinds of rapid sequence determination techniques currently used nowadays.

    De-novo protein sequence analysis is two kinds of rapid sequence determination techniques currently used nowadays. The theory is to create separate groups of radio-labeled oligonucleotides, each of which has a fixed starting point but terminates randomly on a specific or multiple residue. Only by adding several groups of oligonucleotides to several adjacent swim lanes in the sequencing gel, the oligonucleotides of each group can be analyzed by electrophoresis under the condition of differentiating different DNA molecules with only one nucleotide difference in length.

    1. shotgun proteomics: Shotgun proteomics strategy is a method of identifying proteins directly from complex protein mixtures without protein purification.

    Shotgun proteomics strategy is a method of identifying proteins directly from complex protein mixtures without protein purification. Usually, proteins are digested into polypeptides and then sequenced by tandem mass spectrometry. The obtained polypeptide spectral data are used to search the database. SEQUEST software is used to analyze the first-order structure information of peptide fragments, so as to identify proteins. Shotgun proteomics, which is used to identify samples of mixed proteins with low copy, hydrophobic proteins and some polar proteins, has the advantage that two-dimensional gel electrophoresis (2- DE) technology cannot replace.