Peptide mapping is based on the molecular weight of proteins and peptides and the characteristics of amino acid composition, using specific proteolytic enzymesto act on specialpeptide chain site.
Reversed-phase high performance liquid chromatography has become a standard method for protein analysis and characterization, especially for the analysis and characterization of therapeutic drugs.
Reversed-phase chromatography has high resolution and good detection sensitivity, and can provide a lot of information about proteins.
Sometimes, protein is analyzed as a complete molecule, but more often, proteolytic enzymes are used to act on special amino acid residues to break the carbon backbone, thereby breaking the protein into small fragments.
Subsequently, reversed-phase high performance liquid chromatography was used to analyze the peptide fragments produced by the cleavage.
This technique is called peptide map analysis and is a standard protein analysis method.
A large amount of information about the protein can be obtained by analyzing the peptide fragment after the protein cleavage by reversed-phase chromatography.
By comparing the peptide map of the expressed protein with the reference standard protein, the purity of the protein and the accuracy of expression can be obtained. Peptide mapping is usually used as a tool for identification and analysis of protein therapeutics.
Peptide maps can be used to identify protein degradation products, such as asparagine and oxidized methionine that undergo deamidation.
Peptide map can confirm or verify the disulfide bond connection, so as to obtain the tertiary structure and therapeutic effect of protein.
The peptide map can determine the glycosylation (adding carbohydrates) site, providing conditions for detailed identification of the oligosaccharides attached to it.
The peptide map obtained by mass spectrometry provides an advanced method for protein identification, peptide sequence analysis and data confirmation.
In biological proteome research, protease hydrolysates are also used for protein identification and quantitative analysis.
transsexual. To complete the protein hydrolysis within a reasonable time, the protein must be denatured. At high temperature (37°C), place the protein in a chaotropic agent such as 6M guanidine hydrochloride or 8M urea, and treat it in a neutral pH (~7.5) buffer for 30 minutes, and the protein can be denatured.
Reduction of disulfide bonds. Disulfide bonds prevent complete denaturation of the protein.
The disulfide bond can usually be reduced by adding a reducing agent such as dithiothreitol (DTT) at a concentration of ~20 mM during the denaturation process of the protein to be hydrolyzed.
Carboxymethylation of free cysteine. If the reduced cysteine remains free, it may reform the disulfide bond in the wrong way.
To avoid this situation, reagents such as iodoacetic acid with a concentration of ~60mM can be added and treated at 37°C for 30 minutes to methylate free cysteine. The reaction is annealed by 100 mM DTT.
Desalination. When urea or guanidine salt is present in the solution, the hydrolysis reaction cannot proceed because trypsin itself, as a protein, will denature and lose its enzymatic activity.
Urea or guanidine salt must be removed by ion exchange or dialysis or the concentration must be reduced to below 1M.
Trypsin hydrolysis. After desalting, dissolve the protein in a buffer of pH 7.5-8.5 (the highest active pH of trypsin)-Tris or ammonium carbonate, and add a portion of trypsin to 20-100 protein components to be hydrolyzed, and then Process proteins in the temperature range from low to 37°C.
The low temperature treatment time is up to 16 hours. At 37°C, hydrolysis can be completed within 1 to 4 hours, depending on the protein.
If the time, temperature or relative concentration of trypsin is too low, the hydrolysis will be incomplete, and some potential cleavage may not occur, eventually leading to the formation of macromolecular peptides containing lysine or arginine.
If the hydrolysis time, temperature, or concentration of trypsin are too high, trypsin will dissolve itself and produce "autolysates", that is, peptides produced by trypsin hydrolysis, which will cause confusion.
The usual practice is to ignore protein and consider trypsin. Perform chromatographic analysis on the obtained sample under the condition of complete protein hydrolysis to understand the degree of trypsin autolysis and the position of any trypsin autolysis peptide product in the peptide map.
During the development of the trypsin hydrolysis protocol, the hydrolysis time, temperature and relative concentration of trypsin and protein were optimized.
When using peptide maps to determine the location of disulfide bonds, the protein must be hydrolyzed without reducing the disulfide bonds. But when the disulfide bond is not reduced, the hydrolysis rate of many proteins is very slow.
In the absence of reducing agent, if the hydrolysis rate is slow or poor, Lys-C can be used instead of trypsin and hydrolyzed in 4M urea to maintain protein denaturation during hydrolysis.
Surfactants are sometimes used to maintain the protein in the solution during the hydrolysis process, but surfactants will reduce the chromatographic resolution and should be avoided as much as possible.
Trypsin hydrolysis analysis. The peptides produced by proteolysis were analyzed by reversed-phase high performance liquid chromatography, and the mobile phase used a TFA-containing system (see pages 15-17), and eluted with a gradient of acetonitrile with an initial concentration of about 5% (the initial concentration of acetonitrile was less than 5 % May lead to the non-reproducibility of the chromatogram that eluted the peptide earlier), and the acetonitrile concentration gradually increased to 70% (see Figure 31).
The gradient elution time depends on the size of the protein to be hydrolyzed.
Large-molecule proteins produce more peptides than small-molecule proteolysis, so the separation of peptides requires longer elution time.
Peptides produced by hydrolysis of small proteins (less than 20kd) can usually be separated within 45 to 60 minutes.
Large molecular proteins (20-50kd) require a longer elution time, generally 60~120 minutes.
Proteins with a molecular weight greater than 50kd need an elution time of 120 to 180 minutes.
The resolution is best when a flow rate of 1~2 ml/min and an appropriate temperature are used.
C18 reversed-phase column is usually used. A column with a pore size of 100 angstroms or 300 angstroms can be used, and the selectivity is usually different.
Protein modification causes changes in peptide retention time. If the protein is changed due to translation or expression errors, degradation (deamidation, oxidation) or process variation, this change will be reflected in one or more peptides.
Due to the sensitivity of the reverse phase interaction with the peptide, any change in the peptide will result in a change in the retention time of the peptide.
In the example in Figure 32, two decapeptides with a difference of one amino acid, one of which is threonine and the other of serine, have two peaks in the reverse phase HPLC spectrum.
Not only is the difference in one amino acid, but the two amino acids are both hydroxy amino acids. The difference is that a methyl group is added to the side chain of threonine.
This shows that any change in protein will be reflected as a change in peptide, leading to a change in the retention time of the peptide.
The essence of peptide mapping analysis is that reversed-phase HPLC can achieve the separation of peptides with subtle differences.
