The method of separation and purification depends on the extracted tissue material and the nature of the material to be extracted. The commonly used methods for extraction and separation of proteins and peptides include salting out, ultrafiltration, gel filtration, isoelectric precipitation, ion exchange chromatography, affinity chromatography, adsorption chromatography, counter current dissolution and enzymatic hydrolysis. These methods are often combined to separate and purify specific substances. At the same time, these methods are also commonly used in the analysis of protein and peptide substances, such as chromatography, called swimming and so on.

1.1 high performance liquid chromatography (HPLC)

The emergence of HPLC provides a favorable method for peptide separation, because compared with other compounds, the application of HPLC in protein and peptide can not only complete the separation purpose in a short time, but also produce bioactive peptides on a large scale. Therefore, many scholars have done a lot of work to find the best conditions for the separation and preparation of peptides. How to keep the activity of peptide, how to choose the material of stationary phase, the type of eluent and how to analyze and determine are all the research contents at present.

1.1.1 RP-HPLC

Results the relationship between retention value and retention value: the retention of peptides with different structures on the column should be determined first by RP-HPLC. In order to obtain a series of retention coefficients, wilce et al. Analyzed the retention properties and structures of 2106 kinds of peptides by using multiple linear regression method, and obtained the relationship between the retention coefficients of different amino acid compositions, in which the polar amino acid residues in the 2-20 amino acid composition peptide can reduce the retention time on the column;In the peptide composed of 10-60 amino acids, more nonpolar amino acids can also reduce the retention time on the column, while in the small peptide containing 5-25 amino acids, the increase of nonpolar amino acids can prolong the retention time on the column.

At the same time, many literatures reported the influence of peptide chain length, amino acid composition, temperature and other conditions on the retention, and the best conditions for the separation and extraction of each peptide were obtained by computer processing analysis.

Peptide mapping: peptide mapping is based on the molecular weight and amino acid composition of proteins and peptides, using a specific proteolytic enzyme [endopeptidase] to act on a specific peptide site to break the peptide into small pieces, and forming a characteristic fingerprint through certain separation and detection means. Peptide mapping analysis It is of great significance to study the structure of polypeptide and identify its synthetic characteristics.

Based on the properties of peptide chains that trypsin can specifically act on the carboxyl end of Arg and Lys, the characteristic peptide map of recombinant human growth hormone was determined by RP-HPLC with C18 column. At the same time, the peptide map of insulin was also obtained by the specific cleavage of V8 enzyme, and insulin from different genera with only one amino acid disability could be identified. The monoclonal antibody structure of human tumor necrosis factor was also determined by enzymatic method and on-line analysis technology. This technology has been widely used in the development of new drugs.

1.1.2 hydrophobic interaction chromatography (HIC)
HIC uses hydrophobic gene in polypeptide, which can produce hydrophobic interaction with stationary phase to achieve the purpose of separation and analysis. Compared with rp-gplc, HIC has less denaturation of polypeptide. The structure and activity of GH production by GIC are more stable than that by ep-gplc. Geng et al. Used the low denaturation characteristic of HIC column to denaturate the recombinant interferon - γ expressed by Escherichia coli by guanidine hydrochloride. The products with high bioactivity were purified and folded by HIC column. Different human urinary epidermal growth factor (EGF) was also purified by HIC, which had good biological activity. HIC can purify the sample without ion exchange column. But RP-HPLC can not meet this requirement.

1.1.3 molecular exclusion chromatography (SEC)

SEC is to separate and purify polypeptide materials by using the difference of the size and shape of polypeptide molecules. It is more convenient for some larger aggregated molecules, such as the separation of human recombinant growth hormone (hGH). The separation behavior of GH with different structure and configuration is completely different on the SEC column, so it can separate the variants with different configuration or with slight difference in amino acid sequence. The modification is studied by SEC This PEC has the characteristics of long half-life and strong action. Some peptides or proteins with larger molecular weight can be separated and analyzed by this method.

1.1.4 ion exchange chromatography (iexc)

Iexc can be used to separate and purify bioactive peptides under neutral conditions.It can be divided into two categories: cation column and anion column, and some new resins, such as macroporous resin, pore size resin, ion exchange cellulose, dextran gel, agarose gel resin and so on. In the study of peptide separation and analysis, there are many studies on the properties, eluants and elution conditions of peptides. Different conditions of peptide separation are different, especially the ion strength and salt concentration of eluents have great influence on purification. Wu et al. Reported that the conditions for the separation of bovine carbonate isomer, bovine serum albumin and chicken serum albumin proteinase were studied by ion exchange column chromatography, and valuable data were obtained for the separation of these substances in the future.

1.1.5 chromatography of membrane protein (CMP)

The chromatographic system for separating the mixture of strong vegetable water-based protein and polypeptide by CMP + generally has a detergent (such as SDS) to dissolve the membrane protein and form SDS fusion membrane protein, which is separated and purified by the column with hydroxyapatite as the stationary phase. Hydroxyapatite column has both anionic phosphate group (p-terminal) and cationic calcium (C-terminal). The binding of hydroxyapatite column to immobilization is mainly determined by the size of membrane protein and the binding amount of SDS. The separation mechanism of camp was studied by atomic scattering method. It was found that the exchange of SDS molecules, charged amino acids and charged ions in the stationary phase existed on the ion-exchange column after the sample was combined with SDS, so as to achieve the purpose of hierarchical separation.

1.1.6 high performance displacement chromatography (HPDC)
HPDC uses small molecule high efficiency displacement agent to exchange the sample on the chromatographic column, so as to achieve the purpose of separation. It has the characteristics of separating components with less content. The active human recombinant growth hormone (rhG) which was less than 1% of the total amount was identified by HPDC. In the study of nontoxic agent, jayarama found that dextrose sulfate (DS) is a good replacement agent for β lactoglobulin A and B. generally, the molecular weight of DS is 1 × 104 and 4 × 104. The results show that the lower the molecular weight of the displacer is, the easier it is to combine with the immobilization. Therefore, the smaller the displacer is needed to displace and purify the peptide with low molecular weight.

1.1.7 perfusion chromatography (PC)

PC is a kind of chromatographic separation method based on the principle of molecular sieve and high-speed flow of mobile phase. The pore size of stationary phase and the velocity of mobile phase directly affect the separation effect. The experiment shows that it has the characteristics of low input and high output in the process of production and preparation. At present, there are many kinds of PC stationary phases available on the market, which are suitable for peptide separation with different molecular weight.

1.2 affinity chromatography (AC)

AC is a chromatography method that uses the specific affinity between ligands connected to the stationary phase matrix and ligands that can interact with them. Since cuatrecasas proposed the concept of affinity chromatography in 1968, many combinations have been found in the search for specific affinity substances, such as antigen antibody, enzyme catalytic substrate, lectin polysaccharide, oligonucleotide and its complementary chain, etc.
At present, monoclonal antibody or biomimetic ligands are mainly used for peptide separation. These ligands are either natural or synthetic according to their structures. Patel et al. Used a series of affinity columns to separate and purify tissue plasma fibrinogen activator protein polypeptide.

Immobilized metal affinity chromatography (LMAC) is an affinity method developed in recent years. Some metal ions, such as Cu2 +, Ni2 +, Fe3 +, were chelated on the stationary phase matrix. This column can contain Lys, met, ASP, Arg, Tyr, Glu and his polypeptides through the chelating side chain. Especially, the structure containing his-x-x-his in the peptide sequence is most easily combined to the metal ion affinity column, and the purification effect is better. Insulin like growth factor (IGF) and dihydrofolate reductase fusion protein were isolated by this method.

Chaiken et al. Reported another affinity chromatography method, which is produced by antisense DNA expression. It has certain affinity with peptide or protein produced by positive strand DNA expression, such as Arg vasopressin receptor complex, which has been separated by this method. The interaction between DNA, protein and peptide complex is also a common method in biological affinity. The synthetic oligonucleotides are bound to the stationary phase matrix, and the sample protein or polypeptide flows through the column.

1.3 capillary electrophoresis (CE) - separation and analysis method

CE was invented by hjerten at the end of 1960s on the basis of traditional electrophoresis technology. It makes use of small capillary instead of traditional large electrophoresis tank and improves electrophoresis efficiency dozens of times。
This technology has developed rapidly since 1980s, and it is a good tool for biochemical analysts and biochemists to separate and characterize peptides and proteins. CE can be divided into the following types according to the application principle: capillary zone electrophoresis Capillary Zone electrophoresis (CZE), capillary isoelectric focusing electrophoresis (Capillary Isoeletric Focusing, CIEF) capillary electrophoresis (CapillaryGelElectrophoresis, CGE) and micellar electrokinetic capillary chromatography (CIEF).

1.3.1 capillary zone electrophoresis (CZE)

CZE separation of peptides is mainly based on the electrification of compounds in different components, and is more accurate than traditional gel electrophoresis. At present, the main problems existing in CZE separation and analysis of peptide are that natural protein or peptide is easy to react with silanol on capillary silica gel column, which affects peak shape and electrophoresis time. In view of these problems, many scholars have made a lot of experiments to improve, such as adjusting the pH value of battery swimming solution, reducing the polar groups reacting with silanol; improving the composition of capillary column material, aiming at polypeptide Different CZE methods were used to study the separation of five small peptides with nine amino acid residues. The basic conditions for the analysis of small peptides were determined, that is, under the condition of low pH, the buffer solution contained a certain concentration of metal ions such as Zn2 +, etc. at this time, the separation speed was fast and accurate.

1.3.2 capillary isoelectric focusing (CIEF)
Due to the different Pi of different proteins and peptides, in the electrophoresis tank with different pH gradients, they can aggregate and precipitate under the condition of isoelectric pH, and separate from other peptides. CIEF is not widely used in the separation and analysis of mixed peptide substances, mainly used in the separation of peptide isomers from different sources, such as rhG isomers. Because of the instability of the cover on the CIEF column, the method is widely used.

1.3.3 Capillary Gel Electrophoresis (CGE)

CGE is based on the principle of molecular sieve. The proteins or peptides treated by SDS are separated by different molecular shape and weight. At present, there is another kind of non - linear, hydrophobic and polyhydrogel column used for separation and analysis of polypeptide substances. This electrophoresis method is suitable for separating peptides with more hydrophobic side chains. This gel is easy to pour, has long service life and stable properties.

1.3.4 micellar electrokinetic capillary chromatography (MECC)

MECC is based on the principle that some neutral molecules with the same charge can be separated by adding surfactants, such as SDS, into the electrophoresis solution. Especially for some small molecular peptides, anionic and cationic surfactants, they can form micelles with a certain charge, thus obtaining a good separation effect. It has been reported that the inclusion of cyclodextrin and other substances in the electrolyte can selectively interact with the cyclopore of cyclodextrin with the peptide containing hydrophobic structure components, so that the peptide can be separated by hydrophobic interaction.

1.4 systematic application of peptide protein separation engineeringThe above mentioned technology of peptide separation is often combined with each other in practical application, and different separation methods are adopted according to different properties of peptide separation. Especially in the post genomic era, for the in-depth study of proteome, people have improved the means of separating polypeptides and proteins, making comprehensive use of various properties of proteins and polypeptides, adopting the conventional methods of protein peptide extraction, including the above mentioned, and using high-efficiency liquid chromatography, capillary electrophoresis, 2-D electrophoresis and other means to separate cells or tissues as much as possible Many protein polypeptides. In the study of proteomics, the technology of separation and identification of proteins and peptides is one of the methods of separation and analysis. In particular, the development of mass spectrometry technology mentioned below has greatly improved the efficiency of protein peptide analysis and identification.

Analysis method

2.1 mass spectrometry (MS)

MS has been widely used in protein and peptide analysis, especially in the on-line analysis after separation and purification. Continuous flow fast atom bombardment (CF FAB) and electrospray ionization (EIS) are new methods developed in recent years.

2.1.1 continuous flow fast atom bombardment (CF FAB)

Cf-fab is a weak ionization technique, which can ionize peptides or small molecular weight proteins into MH + or (M-H) forms. It is mainly used in the separation and detection of peptides. It has a medium resolution, an accuracy of more than + 0.2amu, and a flow rate of 0.5-1.5 μ L · ml-1. In the determination of mobile phase, 0.5% - 10% matrix such as glycerin and high organic solvent should be added to make the sample sensitive at the detection probe.
Cf-fab is often used in combination with HPLC, CEZ and other methods to achieve the purpose of separation and analysis. Many peptide cf-fab analysis methods have been established and have been well applied. For example, Hideaki used this method to study the tetrapeptide series of L-Pro and l-ala. It is proved that L-Pro is stable in the conformation of small peptide. It is of great significance to connect molecules.

2.1.2 electrospray ionization (EIS)

EIS can produce multivalent ionized proteins or polypeptides, allowing the analysis of proteins with a molecular weight of 1 × 105 and a resolution of 1500-2000 amu. The accuracy is about 0.01%. EIS is more suitable for on-line analysis of proteins with high molecular weight, and needs gasification or organic solvents to make samples sensitive. The combination of EIS and HPLC was successful in the separation and analysis of GH and hemoglobin, which can also be used in combination with CEZ.

2.1.3 matrix associated laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS)

At present, MALDI-TOF is an accurate method to determine the molecular weight in protein identification. It is especially suitable for the determination of the relative molecular weight of mixed protein polypeptide substances, with high sensitivity and resolution. It is a necessary tool for proteomics research. At the same time, the combination of liquid chromatography technology can effectively identify peptide substances. Especially when the mass spectrometry technology of various principles is applied in series, not only the relative molecular mass information of polypeptide can be obtained, but also its sequence structure can be determined. This technology will play a decisive role in the future proteomics research.
2.2 nuclear magnetic resonance (NMR)

Because of the pure digitalization of the map signal, the excessively wide overlapping range (due to the too large relative molecular weight) and the weak nuclear signal, NMR has not been widely used in the analysis of protein and peptide substances. With the application of two-dimensional, three-dimensional and four-dimensional NMR, the development of molecular biology and computer processing technology, NMR has gradually become one of the main methods for the analysis of such substances.

NMR can be used to determine the amino acid sequence and the component content of the quantitative mixture. However, there are still many problems to be solved in the application of protein analysis, such as how to make proteins with large molecular weight have specific shape for quantitative and qualitative analysis, how to reduce the time of data processing and so on. Many scholars are studying these problems. Although it is rarely used in protein analysis, NMR is very useful in the analysis of small peptides containing less than 30 amino acids. It can overcome the shortcomings of protein analysis and achieve the purpose of rapid and accurate analysis.

2.3 other

In addition to the above methods, amino acid composition analysis, amino acid sequence analysis, field analytical mass spectrometry, IR, UV spectrum, CD, circular chromatography, bioassay, radioisotope labeling and Immunology methods have been applied to the result identification, analysis and detection of polypeptide substances.

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