Research progress of separation and analysis of peptide substances This review summarizes the extraction and separation methods of peptide substances in recent years, including the latest application progress of high-performance liquid chromatography, electrophoresis, mass spectrometry and nuclear magnetic resonance methods in the study of peptide substances.
Peptide compounds exist widely in nature, and the study of peptides with certain biological activities has always been a main direction of drug development. The active peptides known in the body are mainly produced or obtained from endocrine glands, organs, secretory cells and body fluids. Cell differentiation, neurohormone transmitter regulation, tumor lesions, and immune regulation in life activities are closely related to the active polypeptide . With the rapid development of modern technology, the means to obtain peptides from natural products has also been continuously improved. The application of some new methods and new ideas. New peptides are constantly being discovered and used in disease prevention and treatment. This article introduces the research progress of the main methods of peptide separation and analysis in recent years.
1 Separation method
Which separation and purification method to take depends on the extracted tissue material and the nature of the substance to be extracted. Commonly used methods for the extraction and separation of proteins and peptides include: salting out, ultrafiltration, gel filtration, isoelectric point precipitation, ion exchange chromatography, affinity chromatography, adsorption chromatography, countercurrent dissolution, and enzymolysis Wait. 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 and swimming.
1.1 High performance liquid chromatography (HPLC)
The emergence of HPLC provides an advantageous method for the separation of peptides, because the HPLC application of proteins and peptides can not only complete the separation in a short time under suitable chromatographic conditions, but more importantly, HPLC It can produce biologically active polypeptides on the scale of preparation. Therefore, many scholars have done a lot of work to find the best conditions for the separation and preparation of peptides. How to maintain the activity of the peptide, how to choose the stationary phase material, the type of eluent, and how to analyze and measure are the contents of the current research.
1.1.1 Reverse phase high performance liquid chromatography (RP-HPLC)
The relationship between the results and the retention value: the separation of peptides by RP-HPLC must first determine the retention of peptides of different structures on the column. In order to obtain a series of retention coefficients, Wilce et al. Used multi-linear regression method to analyze the retention properties and structure of 2106 peptides, and obtained the relationship between the different amino acid composition and the retention coefficient. Amino acid peptides can reduce the retention time on the column; in the peptides composed of 10 to 60 amino acids, more non-polar amino acids can also reduce the retention time on the column, while the small containing 5 to 25 amino acids In peptides, the increase in non-polar amino acids can extend the retention time on the column. At the same time, there are many reports on the influence of peptide chain length, amino acid composition, temperature and other conditions on the retention, and the optimal 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, and uses a specific proteolytic enzyme [generally not endopeptidase] to act on special Peptide chain sites cleave peptides into small fragments and form characteristic fingerprints through certain separation and detection methods. Peptide map analysis is of great significance for the identification of peptide structure and identification of characteristics. Using the property that trypsin can specifically act on the peptide chains of Arg and Lys carboxyl ends, the characteristic pancreatic peptide map of recombinant human growth hormone was detected by RP-HPLC method using C18 column. At the same time, the peptide map of insulin is specifically cleaved by the V8 enzyme, and can be used to identify different species of insulin with a difference of only one amino acid. The structure of the monoclonal antibody of human tumor necrosis factor was also determined by enzymolysis and online analysis technology, which was convenient for identification and analysis. This technology has been widely used in the development of new drugs.
1.1.2 Hydrophobic interaction chromatogrphy (HIC)
HIC utilizes the hydrophobic genes contained in the polypeptide, which can generate hydrophobic interaction with the stationary phase to achieve the purpose of separation and analysis. It has the characteristics of less denaturing the polypeptide than RP-GPLC. The structure and activity of GH products separated by GIC are more stable than those separated by EP-GPLC, and the activity is more stable. Geng et al. Used the low denaturation characteristics of HIC columns to denature guanethidine hydrochloride expressed in E. coli to obtain human recombinant interferon-γ. The product with high biological activity is purified and folded through the HIC column. Different human urine epidermal growth factor (EGF) has also been purified by HIC, and all have good biological activity. HIC can purify samples without ion exchange columns. RP-HPLC cannot meet this requirement.
1.1.3 Sizs-Exclusion chromatogrphy (SEC)
SEC is the separation and purification of peptide substances by using the difference in 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). GH with different structures and configurations is on the SEC column. The upper separation behavior is completely different, so that you can separate variants with different configurations or slight differences in amino acid sequence. Using SEC to study the separation method of modified PEG, this PEC has the characteristics of long half-life and strong effect. Some peptides or proteins with larger molecular weights can be separated and analyzed by this method.
1.1.4 Ion-Exchange chromatography (IEXC)
IEXC can separate and purify biologically active polypeptides under neutral conditions, using the different charging properties of the polypeptides. It can be divided into two categories: cationic column and anionic column, as well as some new resins, such as macroporous resin, homogeneous pore resin, ion exchange cellulose, dextran gel, agarose gel resin, etc. In the study of separation and analysis of peptides, there are many studies on the properties of peptides, eluents and elution conditions. Different peptides have different separation conditions, especially the ionic strength and salt concentration of the eluent. Greater impact. Wu et al. Reported on the use of ion-exchange column chromatography to discuss the extraction conditions for the separation of bovine carbonic anhydride isomers, bovine serum albumin, and chicken serum albumin, and obtained valuable data for future studies on the separation of such substances.
1.1.5 Chromatography of Membrane Protein (CMP)
The CMP + separation chromatography system of strong vegetable water-based protein and peptide mixtures generally has detergents (such as SDS) to dissolve membrane proteins to form SDS-melted membrane proteins, which are separated and purified by a column with hydroxyapatite as the stationary phase. The hydroxyapatite column has an anionic phosphate group (P-terminal) and cationic calcium (C-terminal). The binding to the stationary phase is mainly determined by the size of the membrane protein and the amount of SDS binding. The atomic scattering method was used to study the separation mechanism of cAMP. It was found that after the sample was combined with SDS, there was the exchange between SDS molecules, charged amino acids and charged ions in the stationary phase on the ion exchange column, so as to achieve the purpose of fractionation.
1.1.6 High-Performance Displacement Chromatography (HPDC)
HPDC uses small molecule high-efficiency displacer 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. HPDC was used to identify and isolate the active human recombinant growth hormone (rHG) with a fraction of less than 1%. When studying non-toxic exchangers, Jayarama found that Detran Sulfate (DS) is a good substitute for β-lactoglobulin A and B. Generally, the relative molecular mass of DS is 1 × 104 and 4 × 104. Studies have shown that the lower the relative molecular weight of the displacing agent, the easier it is to bind to the stationary phase. Therefore, when separating peptides with a small relative molecular weight, a smaller displacing agent is required to purify them.
1.1.7 Perfusion Chromatography (PC)
PC is a chromatographic separation method based on the principle of molecular sieve and high-speed flowing mobile phase. The pore size of fixed phase and the velocity of mobile phase directly affect the separation effect. The test proves that it has the characteristics of low input and high output in the production and preparation process. There are many types of PC stationary phases currently available on the market, which are suitable for the separation and use of peptides with different molecular weights.
1.2 Affinity Chromatography (AC)
AC is a chromatographic method that uses specific affinity between a ligand attached to a stationary phase matrix and a ligand that can interact with its specificity to separate substances. 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. . For the separation of peptides, the main use of its monoclonal antibody or biomimetic ligands and its affinity, these ligands are natural, but also artificially synthesized according to their structure. Patel et al. Isolated and purified tissue plasma fibrinogen activator protein peptide using a series of affinity columns.
Immobilized Metal Affinity Chromatography (LMAC) is an affinity method developed in recent years. The stationary phase matrix is ​​chelated with some metal ions, such as Cu2 +, Ni2 +, Fe3 +, etc. This column can be matched to bond chelated side chains containing Lys, Met, Asp, Arg, Tyr, Glu and His polypeptides, especially The structure containing His-XXX-His in the peptide sequence is the easiest to bind to the metal ion affinity column, and the purification effect is better. Among them, Insulin Like Growth Factor (IGF), dihydroleaf reductase fusion protein, etc. are used to isolate products with higher purity by this method.
Chaiken et al. Reported another method of affinity chromatography, using antisense DNA expression, which has a certain affinity for peptides or proteins produced by positive strand DNA expression, such as the Arg vasopressin receptor complex. Separated by this method. The interaction between DNA, protein and peptide complexes is also a commonly used method in biological affinity. The synthetic oligonucleotide is bound to the stationary phase matrix, and the sample protein or polypeptide flows through the column, and the binding can achieve the purpose of separating the specific structure polypeptide.
1.3 Capillary electrophoresis (CE)-Separation analysis method CE was invented by Hjerten on the basis of traditional electrophoresis technology in the late 1960s. It uses small capillaries to replace traditional large electrophoresis tanks, which improves electrophoresis efficiency. Dozens of times. This technology has developed rapidly since the 1980s and is an advantageous tool for biochemical analysts and biochemists to separate and characterize peptides and proteinaceous substances. According to different application principles, CE can be divided into the following types: Capillary Zone Electrophoresis (CZE), Capillary Isoeletric Focusing (CIEF), Capillary Gel Electrophoresis (CGE), and Micellar Capillary Electrolyte Analysis (Micellar Electokinetic Electrophoresis Chromatorgraphy, MECC), etc.
1.3.1 Capillary Zone Electrophoresis (CZE)
CZE separation of peptides is mainly based on the chargeability of compounds in different components, which is more accurate than traditional gel electrophoresis. At present, the main problem in the separation and analysis of peptide materials in CZE is that natural proteins or peptides easily react with silanol on the capillary silica gel column, affecting peak shape and electrophoresis time. Many scholars have done a lot of experiments to improve these problems, such as adjustment The pH value of the battery swimming liquid reduces the polar groups that react with silanol; improve the composition of the capillary column material, and adopt different CZE methods to study the separation of 5 small peptides containing 9 amino acid residues according to the different properties of the peptide. The basic conditions for the analysis of small peptides were determined, that is, under low pH conditions, the buffer contains a certain concentration of metal ions such as Zn2 +, etc. At this time, the separation speed is fast and accurate.
1.3.2 Capillary Isleletric Focusing (CIEF)
Because different proteins and peptides have different isoelectric points (PI), in electrophoresis tanks with different pH gradients, they can be aggregated and precipitated under isoelectric point pH conditions and separated from other peptides. CIEF is not widely used in the separation and analysis of mixed peptide substances. It is mainly used to separate peptide isomers from different sources, such as the separation of different isomers of rHG. The instability of the covering on the CIEF column limits the wide application of this method.
1.3.3 Capillary Gel Electrophoresis (CGE)
CGE is based on the principle of molecular sieves. Proteins or peptides treated with sodium dodecyl sulfonate (SDS) are mainly separated by different molecular shapes and molecular weights during electrophoresis. At present, there is another non-crosslinked, linear, hydrophobic polymer gel column used for the separation and analysis of peptide substances. This electrophoresis method is suitable for the separation of peptides with more hydrophobic side chains. This gel is easy to infuse and has a long service life. Long and stable in nature.
1.3.4 Micellar Electrokinetic Electorphoresis Chromatography (MECC)
The principle of MECC is to add surfactants, such as SDS, into the electrophoresis fluid, so that some neutral molecules with the same charge can be separated. Especially for some small molecule peptides, the application of anionic and cationic surfactants can form micelles with a certain charge, so as to obtain a good separation effect. It has been reported in the literature that by adding cyclodextrin and other substances to the electrolyte, the peptides containing hydrophobic structural components can be used to selectively interact with the ring pores of cyclodextrin, thereby using hydrophobic interaction to separate the peptides.
1.4 The system of peptide protein separation engineering uses the above-mentioned technique of separating peptides. In practical applications, they are often combined with each other. According to the nature of the separated peptides, different separation methods are used. Especially in the post-genomic era, for the in-depth study of proteomics, people continue to improve the means of separating peptides and proteins, comprehensively utilize the various properties of proteins and peptides, using the conventional protein peptide extraction methods including the aforementioned, while using High-performance liquid chromatography, capillary electrophoresis, 2-D electrophoresis and other means can separate as many protein and peptides as possible in cells or tissues. The systematic application of protein and peptide separation and identification techniques in proteomics research is both a means of separation and one of analytical methods in this study. In particular, the development of the mass spectrometry technology mentioned below has greatly improved the efficiency of the analysis and identification of protein peptides.
2 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. The high sensitivity and rapidity of MS are particularly suitable for the analysis and identification of peptide substances. Among them, 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 technology that can ionize peptides or small molecular weight proteins into MH + or (MH) form. It is mainly used for separation and detection of peptides. It has medium resolution, accuracy greater than +0.2 amu, and the flow rate is generally 0.5-1.5 μl · Ml-1. In the measurement, the mobile phase needs to add 0.5% -10% base such as glycerin and high organic solvent components 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 analysis. Many polypeptide cf-FAB analysis methods have been established and have been well applied. For example, Hideaki used this method to study the series of tetrapeptide compounds of L-Pro and L-Ala. It proves that L-Pro is maintaining the stability of small peptide structure. It is important to connect molecules.
2.1.2 Electrospray ionization mass spectrometry (Electrospray Ionozation, EIS)
EIS can produce multivalent ionized proteins or peptides, allowing the analysis of proteins with a relative molecular mass of 1 × 105 with a resolution of 1500-2000 amu. The accuracy is around 0.01%. EIS is more suitable for online analysis of proteins with relatively high molecular mass, and requires gasification or organic solvents to sensitize the sample. The separation and analysis of GH and hemoglobin using EIS and HPLC have been successful, and it can also be used in combination with CEZ.
2.1.3 Matrix-associated laser disso-ciation / ionization time of flight mass spectrmtry (MALDI-TOF MS)
MALDI-TOF is a method for accurate determination of molecular mass in protein identification at present. It is especially suitable for the determination of the relative molecular mass of mixed protein peptides, with high sensitivity and resolution. It is an essential tool for current proteomics research. At the same time combined with liquid chromatography combined technology can efficiently identify peptide substances. Especially when mass spectrometry techniques of various principles are applied in series, not only can the molecular mass information of the peptide be obtained, but also its sequence structure can be determined. This technique will play a decisive role in future proteomics research.
2.2 Nuclear Magnetic Resonance (NMR)
NMR has been used in the analysis of proteins and peptides because of the pure digitalization of spectral signals and the excessive overlapping range (due to the relative molecular mass is too large) and weak nuclear signals. With the application of 2D, 3D and 4D NMR, the development of molecular biology and computer processing technology has made NMR one of the main methods for the analysis of such substances. NMR can be used for analysis such as determining the amino acid sequence and quantifying the composition of each component in the mixture. However, there are still many problems to be solved in protein analysis. For example, how to make proteins with large molecular weight have a specific shape to facilitate quantitative and qualitative analysis, and how to reduce the time of data processing. Many of these issues are being studied by many scholars. Although it is rarely used in protein analysis, NMR is very useful when analyzing small peptides containing less than 30 amino acids in the molecule, which can overcome the shortcomings in protein analysis and achieve the purpose of rapid and accurate analysis.
2.3 In addition to the above methods, amino acid composition analysis, amino acid sequence analysis, field analysis mass spectrometry, IR, UV spectroscopy, CD, circular chromatography, biological identification methods, radioisotope labeling methods and immunological methods have been applied to peptides Substance identification, analysis and testing.
The above briefly introduces the common methods and latest research directions of peptide substance separation and analysis in recent years. With the continuous development of the level of science and technology, there will be many newer separation analysis methods constantly emerging, so research in this field has broad prospects.
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