Prior to detailed peptide/Protein sequence analysis, first introduce Shotgun Protein Identification and PMF analysis:
Shotgun proteomics refers to the study of all proteins in complex mixtures such as serum, urine and cell lysates using bottom-up proteomics techniques. HPLC is used in combination with MS. Membrane protein identification is very important, Membrane Protein such as receptor and ion channel are the key regulatory factors of cell function. Membrane protein accounts for two-thirds of known drug targets, highlighting its important drug importance. G protein coupled receptor (GPCRs) is the largest and most widely used type of membrane receptor and the most important drug, accounting for more than 50% of all human drug targets, and is a therapeutic target for cancer, cardiovascular, metabolic, central nervous system and inflammatory diseases. Ion channels are another group of important drug targets for membrane proteins, accounting for 10% of drug activity on the market. Although membrane receptors are important, it is very difficult to prepare them in a sufficient number of pure, correctly folded forms. By means of Shotgun proteomics, 10 membrane proteins over the membrane domain can be identified. The most remarkable feature of Shotgun proteomics is its ability to simultaneously identify and compare and quantify multiple proteins with minimal protein isolation. Protein mixtures were first digested by protease and the resulting peptides were isolated in HPLC and then sequenced by tandem mass spectrometry/mass spectrometry to determine the sequence of each peptide. The identified peptide sequences were compared with the database to find the corresponding protein markers.
Peptide mass fingerprinting (PMF) is an analytical technique for identifying unknown proteins. The protein to be identified is first digested into a polypeptide whose absolute mass can be accurately analyzed by mass spectrometers such as maltitol or esi-tof.MALDI is preferred in PMF analysis because it has extensive sequence coverage and can analyze large Numbers of samples at the same time. The peptide mass was compared with a database of theoretical peptide mass containing all known protein sequences. The match with the highest score was identified as an unknown protein. Since only the mass of the peptide is measured, but not the exact sequence of the peptide, it will interfere with the mass matching. For example, the quality of the peptide was increased by the translation modification, and the contamination of other proteins also affected the accuracy of the analysis. Therefore, most post-translational modifications and high contamination of other proteins should be eliminated before analysis.
Both of them help us to carry out the sequence analysis of peptide or protein, followed by the explanation of the sequence analysis of peptide or protein:
Sequence analysis of peptides or proteins can be obtained by analyzing therapeutic proteins, monoclonal antibodies and protein vaccines. N-terminal Edman degradation refers to the process of protein n-terminal sequence analysis in which the protein is firstly modified with PITC.Subsequently, the derivatized terminal amino acid is chopped by acid in the form of phenyl thiohydantoin(a prime derivative and a new final-amino group the next amino acid is now available for the next round of reactions by the supplier. Therefore, the protein sequence can be analyzed by adding the severe reaction of PITC and cutting 1 PTH at a time. Assuming that the reaction is completely effective, the method can sequence the entire amino acid from the n-terminal. Although less efficient than mass spectrometry, n - end Edman sequencing has some advantages over other methods in protein analysis. Using current techniques, it is fairly routine to obtain at least 20 to 25 sequence residues from the n-terminal of proteins and peptides.
In addition, the most commonly used protein sequence analysis technique is mass spectrometry. Mass spectrometry (ms) is an efficient method for accurate determination of protein mass and characterization of proteins. The two main mass spectra are electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI).Basically, proteins are digested into small fragments after being pretreated by digestive enzymes and then analyzed using a mass spectrometer. According to the needs of the project, mardi and ESI are ionization sources of two ionizing peptide fragments. Typically, MALDI is more commonly used when dealing with large Numbers of samples. Then the peptide sequences were obtained by analyzing the mass spectra of each fragment, which together formed a complete protein sequence.
The peptide/protein sequence is of great significance for studying the biological function of peptide/protein. However, the full characterization of peptides/proteins, including post-translational modifications (PTMs), sequence mutations and mutations, is very challenging. There are two methods to determine peptide/protein sequences by mass spectrometry: database search and DE novo sequencing. Database retrieval methods compare the mass spectra obtained with the database of known protein sequences to determine the protein sequences.DE novo sequencing is a process of interpreting amino acid sequences directly from a tandem mass spectrum without a database. Although the mass spectrometry method is well established for protein database retrieval and identification, it is not applicable if there is no protein sequence in the database at present. Therefore, de novo sequencing is the only way to identify new peptides, sequenced organisms, antibody drugs, and database search methods that cannot be detected.
Science and technology are constantly improving, and it is believed that the sequence analysis method of peptide or protein will develop more easily!
