1 separation method
What kind of separation and purification method is adopted depends on the nature of the extracted tissue material and the substance to be extracted. Common methods for extracting and separating protein and peptides include salting-out, ultrafiltration, gel filtration, isoelectric precipitation, ion exchange chromatography, affinity chromatography, adsorption chromatography, countercurrent fractionation, enzymatic hydrolysis, etc. These methods are often combined to separate and purify specific substances. These methods are also commonly used in protein and peptide analysis, such as chromatography and swimming.
1. 1 HPLC
The appearance of high performance liquid chromatography provides a favorable method for the separation of peptides, because compared with other compounds, protein and peptides can not only be separated in a short time under suitable chromatographic conditions, but more importantly, bioactive peptides can be produced on a preparation scale by high performance liquid chromatography. 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 peptides, how to choose stationary phase materials, the types of eluents and how to analyze and determine them are all the contents of current research.
1. 1. 1 RP-HPLC
Relationship between results and retention values: To separate peptides by RP-HPLC, the retention values of peptides with different structures on the chromatographic column should be determined first. In order to obtain a series of retention coefficients, Wilce et al. analyzed the retention properties and structures of 265,438+006 peptides by multiple linear regression method, and obtained the relationship between different amino acid compositions and retention coefficients. Among them, the polar amino acid residues in the peptide segment composed of 2 ~ 20 amino acids can reduce the retention time on the column; In the small 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 effects of peptide chain length, amino acid composition, temperature and other conditions on the retention. Through computer processing and analysis, the best conditions for the separation and extraction of each polypeptide were obtained.
Peptide map: According to the characteristics of protein, molecular weight and amino acid composition of polypeptide, peptide map uses a highly specific proteolytic enzyme [usually endopeptidase] to act on a specific peptide chain site to split the polypeptide into small fragments, and forms a characteristic fingerprint through certain separation and detection means. Peptide map is of great significance to the study of polypeptide structure and the identification of its characteristics. Based on the characteristic that trypsin can specifically act on the carboxyl-terminal peptide chain of Arg and Lys, the characteristic peptide map of recombinant human growth hormone was detected by reversed-phase high performance liquid chromatography with C 18 column. At the same time, the peptide map of insulin is also prepared by V8 enzyme specific cleavage, which can identify different kinds of insulin with only one amino acid difference. The structure of monoclonal antibody against human tumor necrosis factor was also determined by enzymolysis and on-line 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 chromatography (HIC)
HIC uses hydrophobic gene contained in polypeptide to interact with stationary phase to achieve the purpose of separation and analysis, and has the characteristic that the denaturation of polypeptide is smaller than that of RP-GPLC. The structure and activity of GH products separated by GIC are more stable than those separated by EP-GPLC. Geng et al. used the low denaturation characteristics of HIC column to denature the expression of Escherichia coli by guanethidine hydrochloride to obtain human recombinant interferon-γ. The products with high bioactivity were purified and folded by HIC column. Different human urinary epidermal growth factors (EGF) were also purified by HIC, and all of them had good biological activities. HIC can purify the sample without ion exchange column. But RP-HPLC can't meet this requirement.
1. 1.3 molecular size exclusion chromatography (sec)
SEC is to separate and purify polypeptide substances by using the differences in the size and shape of polypeptide molecules, especially for some large aggregation molecules, such as human recombinant growth hormone (hgH). The separation behavior of GHs with different structures and configurations on SEC column is completely different, so variants with different configurations or slightly different amino acid sequences can be separated. The separation method of modified PEG was studied by SEC. The PEC has the characteristics of long half-life and strong action. Some peptides or protein with large molecular weight can be separated and analyzed by this method.
1. 1.4 ion exchange chromatography (IEXC)
IEXC can use different charges of peptides to separate and purify bioactive peptides under neutral conditions. It can be divided into cation column and anion column, and there are some new resins, such as macroporous resin, homogeneous resin, ion exchange cellulose, dextran gel, agarose gel resin and so on. In the separation and analysis of peptides, there are many studies on the properties of peptides, eluents and elution conditions, and different separation conditions are also different, especially the ionic strength and salt concentration of eluents have great influence on purification. Wu et al reported the extraction conditions of separating bovine carbonic anhydride isomers, bovine serum albumin and chicken serum protease by ion exchange column chromatography, which obtained valuable data for the separation of such substances in the future.
1. 1.5 membrane protein (CMP) chromatography
In the CMP+ separation chromatography system of the mixture of protein and polypeptide in strong vegetables, membrane proteins are generally dissolved by detergents (such as SDS) to form SDS fusion membrane proteins, which are separated and purified by a column with hydroxyapatite as the stationary phase. Hydroxyapatite column has both anionic phosphate group (P-terminal) and cationic calcium (C-terminal), and the binding with stationary phase is mainly determined by the size of membrane protein and the amount of SDS binding. The separation mechanism of cAMP was studied by atomic scattering method. It was found that SDS molecules, charged amino acids and charged ions were exchanged in the stationary phase on the ion exchange column after the sample was combined with SDS, thus achieving the purpose of classification and separation.
1. 1.6 high performance displacement chromatography (HPDC)
HPDC is to exchange samples on the chromatographic column by using small molecule high efficiency displacer, so as to achieve the purpose of separation. It has the characteristic of separating components with less content. Active recombinant human growth hormone (rHG) was isolated from HPDC, accounting for 65438 0% of the total. Jayarama found that Diethylene Sulfate (DS) is a good substitute for β lactoglobulin A and B, and the relative molecular weight of DS is generally 1× 104 and 4× 104. The research shows that the lower the relative molecular weight of the displacer, the easier it is to combine with the immobilization. Therefore, when peptides with low relative molecular weight are separated, a smaller displacer is needed to displace and purify them.
1. 1.7 perfusion chromatography (PC)
PC is a chromatographic separation method based on molecular sieve principle and high-speed mobile phase. The pore size of stationary phase and the velocity of mobile phase directly affect the separation effect. Experiments show that it has the characteristics of low input and high output in the production and preparation process. At present, there are many PC stationary phases available in 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, which separates substances by using the specific affinity between ligands attached to stationary phase matrix and ligands that can interact with them specifically. Since Cuatrecasas put forward the concept of affinity chromatography in 1968, many combinations have been found in the process of finding specific affinity substances, such as antigen-antibody, enzyme catalytic substrate, lectin-polysaccharide, oligonucleotide and its complementary chain. At present, monoclonal antibodies or biomimetic ligands are mainly used for the separation of polypeptides, and these ligands are either naturally synthesized or artificially synthesized according to their structures. Patel et al. separated and purified tissue plasma plasminogen activator protein polypeptide through a series of affinity columns.
Immobilized metal affinity chromatography (LMAC) is an affinity method developed in recent years. Some metal ions, such as Cu2+, Ni2+, Fe3+, etc. Chelated on the stationary phase matrix. This column can chelate polypeptides with side chains containing Lys, Met, Asp, Arg, Tyr, Glu and his, especially the structure with His-X-X-X-His in the peptide sequence, which is the easiest to combine with metal ion affinity column and has good purification effect. Among them, the fusion protein of insulin-like growth factor (IGF) and dihydroleaf reductase was separated into high-purity products by this method.
Chaiken et al. reported another affinity chromatography method, which is produced by antisense DNA expression and 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 of DNA with protein and polypeptide complexes is also a common method in biological affinity. The synthesized oligonucleotide is bound to the stationary phase matrix, and the sample protein or polypeptide flows through the column, and the polypeptide with specific structure can be separated.
Capillary Electrophoresis (CE)-Separation and Analysis Methods
CE was invented by Hjerten in the late 1960s on the basis of traditional electrophoresis technology. It uses small capillary instead of traditional large electrophoresis tank, which improves electrophoresis efficiency by dozens of times. This technique has developed rapidly since 1980s, and it is a favorable tool for biochemical analysts and biochemists to separate and characterize peptides and protein. According to different application principles, CE can be divided into the following categories: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary gel electrophoresis (CGE) and micellar electrokinetic capillary chromatography (MECC).
Capillary zone electrophoresis (CZE) 1. 3. 1
The separation of peptides by CZE is mainly determined by the electrification of compounds with different components, which is more accurate than traditional gel electrophoresis. At present, the main problem in CZE separation and analysis of polypeptide substances is that natural protein or polypeptide can easily react with silanol on capillary silica gel column, which affects the peak shape and electrophoresis time. Many scholars have done a lot of experiments to improve these problems, such as adjusting the PH value of battery electrophoresis solution and reducing the polar groups that react with silanol; By improving the composition of capillary column materials, according to the different properties of peptides, five kinds of small peptides with nine amino acid residues were separated by different CZE methods, and the basic conditions for the analysis of small peptides were determined, that is, at low PH, the buffer solution contained a certain concentration of metal ions such as Zn2+, and the separation speed was fast and accurate.
1.3.2 capillary electrophoresis focusing (CIEF)
Because different protein and peptides have different isoelectric points (PI), they can be aggregated and precipitated in electrophoresis tanks with different pH gradients and separated from other peptides. CIEF is not widely used in the separation and analysis of mixed polypeptide substances, but mainly used for the separation of polypeptide isomers from different sources, such as the separation of different isomers of rHG. The instability of the surface coating of CIEF column limits the wide application of this method.
1.3.3 Capillary gel electrophoresis (CGE)
CGE is based on the principle of molecular sieve. Protein or peptides treated with sodium dodecyl sulfonate (SDS) were separated by different molecular shapes and molecular weights during electrophoresis. At present, another non-crosslinked, linear and hydrophobic polymer gel column is used to separate and analyze polypeptide substances. This electrophoresis method is suitable for the separation of peptides with more hydrophobic side chains. This gel is easy to pour, long in service life and stable in performance.
1.3.4 micellar electrokinetic capillary chromatography (MECC)
The principle of MECC is to add surfactants, such as SDS, to the electrophoresis solution, so that some neutral molecules with the same charge can be separated. Especially for some small molecular peptides, the application of anionic and cationic surfactants can make them form micelles with certain charges, thus obtaining good separation effect. It has been reported that when cyclodextrin and other substances are added to the electrolyte, peptides containing hydrophobic structural components can selectively interact with the ring holes of cyclodextrin, thus separating peptides through hydrophobic interaction.
Systematic application of 1.4 polypeptide protein separation project.
The above-mentioned techniques for separating polypeptides are often combined in practical applications, and different separation methods are adopted according to the different properties of separated polypeptides. Especially in the post-genome era, in order to further study the protein Group, people have continuously improved the means of separating peptides and protein, comprehensively utilized the properties of protein and peptides, adopted the above-mentioned conventional protein polypeptide extraction method, and used high performance liquid chromatography, capillary electrophoresis, two-dimensional electrophoresis and other means to separate as many protein peptides from cells or tissues as possible. The systematic application of protein and polypeptide isolation and identification technology in protein omics research is not only a separation method, but also an analysis method. Especially, the development of mass spectrometry mentioned below has greatly improved the analysis and identification efficiency of protein peptides.
2 analysis methods
2. 1 MS)
Mass spectrometry has been widely used in protein and polypeptide analysis, especially on-line analysis after separation and purification. Its high sensitivity and rapidity are particularly suitable for the analysis and identification of polypeptide 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 technique, which can ionize peptides or small molecular weight protein into MH+ or (M-H) forms. It is mainly used for the separation and detection of peptide fragments, with medium resolution and accuracy greater than +0.2amu, and the flow rate is generally 0.5- 1.5 μ l mL- 1. During the determination, 0.5%- 10% matrix such as glycerol and high organic solvent should be added to the mobile phase to sensitize the sample at the detection probe. Cf-FAB is often combined with HPLC, CEZ and other methods to achieve the purpose of separation and analysis. Cf-FAB analysis methods of many peptides have been established and well applied. For example, Hideaki and others studied the tetrapeptide series of L-Pro and L-Ala in this way. It is proved that L-Pro can maintain the phase stability of small peptides. It is of great significance in connecting molecules.
2. 1.2 electrospray ionization mass spectrometry (EIS)
EIS can produce multivalent ionized protein or polypeptide, allowing the analysis of protein with relative molecular weight of 1× 105 and resolution of 1500-2000amu. The accuracy is about 0.0 1%. EIS is more suitable for on-line analysis of protein with relatively large molecular weight, which requires gasification or organic solvent to sensitize the sample. Growth hormone and hemoglobin were successfully separated and analyzed by electrochemical impedance spectroscopy and high performance liquid chromatography, and can be combined with CEZ.
2. 1.3 matrix-related laser desorption ionization/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
MALDI-TOF is an accurate method to determine molecular weight in protein identification at present, especially suitable for the determination of relative molecular weight of mixed protein and polypeptide substances with high sensitivity and high resolution. It is an essential tool for protein omics research at present. At the same time, combined with liquid chromatography, peptides can be identified efficiently. Especially, mass spectrometry with various principles in series can not only obtain the relative molecular weight information of peptides, but also determine their sequence structure, which will play a decisive role in the future protein omics research.
2.2 nuclear magnetic resonance (NMR)
Due to the pure digitization of spectral signals, too wide overlapping range (due to too large relative molecular weight) and weak nuclear signals, NMR is not widely used in the analysis of protein and peptides. With the application of two-dimensional, three-dimensional and four-dimensional nuclear magnetic resonance, the development of molecular biology and computer processing technology, nuclear magnetic resonance has gradually become one of the main methods to analyze such substances. Nuclear magnetic resonance can be used to determine the amino acid sequence and quantify the composition of each component in the mixture. However, there are still many problems to be solved in protein analysis, such as how to make the protein with large molecular weight have a specific shape for quantitative and qualitative analysis, and how to reduce the time of data processing. Many scholars are studying these problems. Although nuclear magnetic resonance is rarely used in protein analysis, it is very useful in analyzing small peptides with less than 30 amino acids in molecules, which can overcome the shortcomings of protein analysis and achieve the purpose of rapid and accurate analysis.
2.3 Others
In addition to the above methods, amino acid composition analysis, amino acid sequence analysis, field analysis mass spectrometry, IR, UV spectrum, CD, ring chromatography, biological identification, radioisotope labeling and immunological methods have all been applied to the identification, analysis and detection of polypeptide substances.
This paper briefly introduces the commonly used methods and the latest research direction of separation and analysis of polypeptide substances in recent years. With the continuous development of science and technology, many newer separation and analysis methods will appear, so the research in this field has broad prospects.
Application of SDS-PAGE in displaying small molecular peptides
SDS-PAGE is widely used in the separation, identification and purification of protein. The effective separation range depends on the concentration and crosslinking degree of polyacrylamide. Its pore size decreases with the increase of the ratio of bisacrylamide to acrylamide, and reaches the minimum when it approaches 1: 20. Small molecular peptides with molecular weight lower than 10kD can't be completely separated by SDS-PAGE with high concentration polyacrylamide gel, or they are not excellent or weak and dispersed. And the smaller the molecular weight, the worse the effect.
In order to display small molecular weight peptides on SDS-PAGE, two methods are usually used: one is to increase the concentration and crosslinking degree of the gel, add some solute molecules that can reduce the pore size of polyacrylamide gel, and use substances such as urea, glycerol or sucrose; The second is to select the kind and concentration of trailing ions in the buffer to improve the separation effect of peptides.
operation sequence/order
1. The preparation of electrophoresis buffer is shown in the following table.
Buffer Tris
Triazine (mol/L)
(mol/L) Sodium dodecyl sulfate
(%)
Anode buffer solution
Cathode buffer solution
Glue buffer 0.2
0. 1
3.0—
0. 1
—8.9*
8.25**
8.4*—
0. 1
0.3
* adjust the pH value with hydrochloric acid.
* * The value is about 8.25.
2. Preparation of acrylamide storage solution
Monopropyl and Dipropyl Percentage of Monopropyl-Dipropyl Mixture
49.5% T，3%C
49.5% T，6%C48
46.5 1.5
3.0
T: total concentration of acrylamide.
C: degree of crosslinking
3. Preparation of glue, similar to general SDS-PAGE, separation glue and concentrated glue are prepared according to the following table.
Component separation glue
16% T, 6%C concentrated glue
6% T，3%C
49.5% T, 3%C acrylamide solution (ml)
49.5% T, 6%C acrylamide solution (ml)
Gel buffer (ml)
Urea (g) [Glycerol (ml)]
Water (ml)
10% ammonium persulfate (microliter)
TEMED (microliter)
Total volume (ml)
3.3
3.3
3.6[2.4]
1
40
4.0
10.040.48
—
1.00
—
1.50
25
2.5
3.03
4. Sample buffer
4% sodium dodecyl sulfate
12% glycerol
50 mmol/L trihydroxymethyl aminomethane
2% mercaptoethanol
0.0 1% Serva blue
Mix the polypeptide sample with the sample buffer and boil it for 2 minutes (or take a water bath at 40℃ for 30 minutes).
5. Fix the glass plate filled with glue on the electrophoresis device, seal the edge with 1% agarose, pour the cathode buffer, and add the samples in turn.
6. Put the electrophoresis apparatus into the electrophoresis tank, pour the anode buffer solution, connect the anode and cathode with the electrophoresis apparatus, and keep the constant voltage at 50~60V. After the indicator enters the separation gel, the voltage can be increased to 70~90V, and the electrophoresis will stop at constant pressure for about 3 hours.
7. Dyeing, decoloring and glue preservation are the same as SDS-PAGE.