Research progress on separation and analysis methods of peptide materials

This paper reviews the methods of extraction, separation and analysis of peptides in recent years, including the latest application progress of high performance liquid chromatography, electrophoresis, mass spectrometry and nuclear magnetic resonance in the study of peptides.

Polypeptide compounds are widely found in nature, and research on polypeptides with certain biological activities has been a major direction of drug development. The active polypeptides known in vivo are mainly produced or obtained from endocrine gland tissues, secretory cells and body fluids. Cell differentiation, neurohormonal neurotransmitter regulation, tumor pathology and immunomodulation in life activities are closely related to active polypeptides. . With the rapid development of modern technology, the means of obtaining peptides from natural products has also been continuously improved. With the application of some new methods and new ideas, new peptides have been found to be used in disease prevention and treatment. This paper introduces the research progress of the main methods for the separation and analysis of peptides in recent years. 1 Separation method The separation and purification method is determined by the nature of the extracted tissue material and the substance to be extracted. Common methods for protein and peptide extraction and separation include: salting out, ultrafiltration, gel filtration, isoelectric precipitation, ion exchange chromatography, affinity chromatography, adsorption chromatography, countercurrent fractionation, enzymatic hydrolysis Etc [1]. These methods are often combined to separate and purify specific substances, and these methods are also commonly used in the analysis of proteins and peptides, such as chromatography, electrophoresis, and the like.

1.1 High Performance Liquid Chromatography (HPLC)
The appearance 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 the appropriate chromatographic conditions, but more importantly, HPLC. Bioactive polypeptides can be produced on a preparative scale. Therefore, many scholars have done a lot of work in finding 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, how to analyze and determine is the current research content.

1.1.1 Reversed Phase High Performance Liquid Chromatography (RP-HPLC)
Relationship between results and retention values: The separation of polypeptides by RP-HPLC first determined the retention of peptides of different structures on the column. In order to obtain a series of retention coefficients, Wilce et al [2] used multi-linear regression method to analyze the retention properties and structure of 2106 peptides, and obtained the relationship between different amino acid composition on retention coefficient, in which polar amino acid residues are In the peptide of 2 to 20 amino acids, the retention time on the column can be reduced; in the peptide of 10 to 60 amino acids, more non-polar amino acids can also reduce the retention time on the column. In small peptides containing 5 to 25 amino acids, an increase in non-polar amino acids can extend the retention time on the column. At the same time, many literatures have reported the effects of peptide chain length, amino acid composition, temperature and other conditions on retention, and computer processing analysis to obtain the best conditions for the separation and extraction of each peptide [3].
Peptide Mapping: Peptide mapping is based on the molecular weight and amino acid composition of proteins, peptides, and the use of specific proteolytic enzymes [generally endopeptidase] for specific The peptide chain site cleaves the polypeptide into small fragments, and a characteristic fingerprint is formed by certain separation detection means. Peptide mapping analysis is of great significance for the study of peptide structure and identification of properties. The peptide fragment of recombinant human growth hormone (rhGH) was detected by RP-HPLC on the C18 column by using the trypsin-specific peptide chain at the carboxyl terminus of Arg and Lys. The characteristic tryptic peptide map of human growth hormone was successfully obtained. [4]. At the same time, the peptide map of insulin was also prepared by specific cleavage of V8 enzyme, and it was possible to identify insulin of different species originating only one amino acid disability [5]. The monoclonal antibody structure of human tumor necrosis factor was also determined by enzymatic hydrolysis and on-line analysis techniques to facilitate the identification and analysis. This technology has been widely used in the development of new drugs.

1.1.2 Hydrophobic interaction chromatography (HIC)
HIC utilizes a hydrophobic group containing a hydrophobic group to create a hydrophobic interaction with the stationary phase for separation analysis, which has less characteristics of denaturation of the polypeptide than RP-HPLC. The structure and activity of the production of hormone (GH) products by HIC separation are more stable than those separated by RP-HPLC, and the activity is relatively stable. Geng et al [6] used the low-denatured characteristics of HIC column to denature transgenic E. coli to obtain human recombinant interferon-γ, and purified and folded high bioactive products through HIC column. Different human urine epidermal growth factor (EGF) was also purified by HIC, and both have good biological activity. HIC can purify samples without ion exchange columns. RP-HPLC does not meet this requirement.

1.1.3 Size-Exclusion chromatography (SEC)
SEC is the use of differences in the size and shape of polypeptide molecules to separate and purify peptide materials, especially for some larger aggregated molecules. For example, in the isolation of human recombinant growth hormone (hGH), the separation behavior of GHs of different structures and configurations on the SEC column is completely different, so that variants with different configurations or slightly different amino acid sequences can be isolated. The separation method of modified PEG was studied by SEC. This PEG has the characteristics of long half-life and strong action [7]. 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 different neutral conditions using the chargeability of the polypeptide. It can be divided into two major categories of cationic column and anionic column, as well as some new resins, such as macroporous resin, uniform pore resin, ion exchange cellulose, dextran gel, agarose gel resin and the like. In the separation and analysis of peptides, the properties of the peptides, eluents and elution conditions are studied. Different peptide separation conditions are different, especially the ionic strength and salt concentration of the eluent. Greater impact. Wu et al [8] reported the extraction conditions of bovine carbonic anhydrase isomers, bovine serum albumin and chicken serum albumin by ion exchange column chromatography, and obtained valuable data for future separation of such substances.

1.1.5 Chromatography of Membrane Protein (CMP)
CMP+ is a chromatographic system for separating highly hydrophobic proteins and peptide mixtures. Generally, detergents (such as SDS) dissolve membrane proteins to form SDS-melt membrane proteins, and are separated and purified from hydroxyapatite as a stationary phase column. The hydroxyapatite column has an anionic phosphate group (P-terminal) and a 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 separation mechanism of cAMP was studied by atomic scattering method. It was found that the sample was combined with SDS and there was exchange of SDS molecules, charged amino acids and charged ions in the stationary phase on the ion exchange column, thus achieving the purpose of fractionation [9].

1.1.6 High-Performance Displacement Chromatography (HPDC)
HPDC uses small molecule high-efficiency displacers to exchange samples on the column for separation purposes. It has the property of separating components with a small amount of components. Active human recombinant growth hormone (rHG) was isolated using HPDC identification below a total of 1% fraction [10]. In the study of non-toxic exchangers, Jayarama [11] found that Detran Sulfate (DS) is a good displacer for β-lactoglobulin A and B. The relative molecular mass of DS is generally 1×104 and 4. ×104 is the most suitable. Studies have shown that the lower the relative molecular mass of the displacer, the easier it is to bind to the stationary phase. Therefore, when separating a polypeptide with a relatively small molecular mass, a smaller displacer is needed to replace it.

1.1.7 Perfusion Chromatography (PC)
PC is a chromatographic separation method based on molecular sieve principle and high-speed mobile mobile phase. The pore size of the stationary phase and the mobile phase velocity directly affect the separation effect. The test proves that it has low input and high output characteristics in the production and preparation process [12]. There are many types of PC stationary phases available on the market, which are suitable for the separation of polypeptides of different molecular weights.

1.2 Affinity Chromatography (AC)
AC is a chromatographic method in which a substance is separated by a specific affinity between a ligand attached to a stationary phase substrate and a ligand which can specifically act. 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 antibodies or biomolecules are mainly used for their separation. These ligands are natural and artificially synthesized according to their structures. Patel et al [13] used a series of affinity columns to separate and purify tissue plasma fibrinogen activator protein peptides.
Immobilized Metal Affinity Chromatography (IMAC) is an affinity method developed in recent years. The stationary phase substrate is compounded with some metal ions, such as Cu2+, Ni2+, Fe3+, etc., and the column can be coupled with a polypeptide containing Lys, Met, Asp, Arg, Tyr, Glu and His in a side chain, especially The structure containing His-XXX-His in the peptide sequence is most easily bound to the metal ion affinity column, and the purification effect is better [14]. Insulin-Like Growth Factor (IGF) and dihydrofolate reductase fusion proteins were isolated by this method.
Chaiken et al [15] reported another affinity chromatography method using an antisense polypeptide as a ligand, which is produced by antisense DNA expression, and has a certain peptide or protein produced by positive-strand DNA expression. Affinity, such as the Arg vasopressin receptor complex, has been isolated by this method. The interaction between DNA and proteins and peptide complexes is also a common method in biological affinity. The artificially synthesized oligonucleotide is bound to the stationary phase substrate, and the sample protein or polypeptide is passed through the column, and combined with it, the purpose of isolating the specific structural polypeptide can be achieved.

1.3 Capillary electrophoresis (CE) - Separation analysis method CE was invented by Hjerten in the late 1960s based on traditional electrophoresis technology. It uses small capillaries instead of traditional large electrophoresis tanks to improve electrophoresis efficiency. Dozens of times. This technology has developed rapidly since the 1980s and is a useful tool for biochemical analysts and biochemists to separate and characterize peptides and proteinaceous materials. CE can be divided into the following according to the application principle; Capillary Zone Electrophoresis (CZE), Capillary Isoeletric Focusing (CIEF), Capillary Gel Electrophoresis (CGE) and Glue Micellar Electrokinetic Electrophoresis Chromatography (MECC) and the like.

1.3.1 Capillary Zone Electrophoresis (CZE)
The separation of peptides by CZE is mainly based on the charge of the compounds in different components, and the separation effect is determined only by the chargeability, which is more accurate than the traditional gel electrophoresis. The main problem currently existing in CZE separation and analysis of peptide materials is that natural proteins or peptides easily react with silanol on the silica gel column of the straw, affecting the peak shape and electrophoresis time. Many scholars have done a lot of experiments to improve these problems, such as The pH of the electrophoresis solution is adjusted to reduce the polar groups reactive with silanol; the composition of the capillary column material is improved, and different CZE columns are used for separation depending on the nature of the polypeptide. Issaq [16] used the CZE method to isolate five small peptides containing 9 amino acid residues, and determined the basic conditions for the analysis of small peptides. Under low pH conditions, the buffer contains a certain concentration of metal ions such as Zn2+. At this time, the separation speed is fast and accurate.

1.3.2 Capillary Isoeletric Focusing (CIEF)
Due to the different isoelectric points (PI) of different proteins and polypeptides, in an electrophoresis tank with different pH gradients, it can be aggregated and precipitated at isoelectric point pH, and separated from other peptides. CIEF is not widely used in the separation and analysis of mixed peptide materials. It mainly uses separation between polypeptide isomers from different sources, such as separation of different isomers of rHG [17]. The wide application of this method is limited by the instability of the surface covering on the CIEF column.

1.3.3 Capillary Gel Electrophoresis (CGE)
CGE is based on the molecular sieve principle. Proteins or peptides treated with sodium dodecyl sulfate (SDS) are mainly separated by molecular shape and molecular weight during electrophoresis. At present, another non-crosslinked, linear, hydrophobic polygel column is used for the separation and analysis of polypeptides. This electrophoresis method is suitable for peptide separation with more hydrophobic side chains. This gel is easy to infuse, has a long service life and is relatively stable in nature.

1.3.4 Micellar Electrokinetic Electrophoresis Chromatography (MECC)
The principle of MECC is to add a surfactant, such as SDS, to the electrophoresis fluid to separate some neutral molecules with the same charge molecules. Especially for the application of some small molecular peptides, anionic and cationic surfactants, it can form a micelle with a certain charge, so as to obtain a good separation effect. It has been reported in the literature that the addition of a cyclodextrin or the like to the electrolyte allows the polypeptide containing the hydrophobic structural component to selectively interact with the ring hole of the cyclodextrin, thereby separating the polypeptide by hydrophobic action [18].

1.4 Systematic application of peptide and protein separation engineering The above-mentioned techniques for isolating polypeptides are combined with each other in practical applications, and different separation means are employed depending on the nature of the isolated polypeptide. Especially in the post-genome era, for the in-depth study of the proteome [19], the methods for isolating peptides and proteins have been continuously improved, and various properties of proteins and peptides have been comprehensively utilized, including the conventional protein peptide extraction method mentioned above. At the same time, high-performance liquid chromatography, capillary electrophoresis, 2-D electrophoresis and the like are used to isolate as many protein polypeptides as possible in cells or tissues. The systematic application of protein and peptide separation and identification techniques in proteomics research is the key to achieving proteome planning. Among them, electrophoresis technology is one of the separation methods and one analysis method in this research. In particular, the development of the mass spectrometry technology mentioned below greatly improves the efficiency of analysis and identification of protein polypeptides.

2 Analytical methods

2.1 Mass Spectrometry (MS)
MS has been widely used in protein and peptide analysis, especially in the online 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 technique that ionizes peptides or small molecular weight proteins into MH+ or (MH) forms. It is mainly used for the separation and detection of peptides. It has medium resolution with an accuracy of more than ±0.2 amu and a flow rate of generally 0.5-15 μl.mL-1. In the measurement, the mobile phase needs to add 0.5% to 10% of matrix such as glycerin and high organic solvent components to make the sample sensitized at the detection probe [20]. cf-FAB is often used in combination with HPLC, CEZ and other methods for separation analysis. The cf-FAB analysis method for many peptides has been established and is well applied. For example, Hideaki et al. used this method to study the series of tetrapeptide compounds of L-Pro and L-Ala. It is proved that L-Pro plays an important role in maintaining the stability of small peptides and linking molecules [21].

2.1.2 Electrospray ionization mass spectrometry (EIS)
EIS can produce multivalent ionized proteins or peptides, allowing analysis of proteins with a molecular mass of 1×105, with resolutions ranging from 1500 to 2000 amu and accuracy of around 0.01%. EIS is more suitable for on-line analysis of proteins with relatively high molecular mass and requires gasification or organic solvents to sensitize the sample. The combination of EIS and HPLC for the analysis of GH and hemoglobin was successful, and it can also be combined with CEZ [22].

2.1.3 Matrix-associated laser dissociation/ionization time of flight mass spectrometry (MALDI-TOF MS)
MALDI-TOF is a means for accurately determining the relative molecular mass in protein identification. It is especially suitable for the determination of the relative molecular mass of mixed protein peptides with high sensitivity and resolution. It is an indispensable tool for current proteomics research. At the same time, combined with liquid chromatography combined technology can efficiently identify peptide substances. Especially when the mass spectrometry techniques of various principles are applied in series, not only can the relative molecular mass information of the polypeptide be obtained, but also its sequence structure, which will play a decisive role in future proteomics research [23].

2.2 Nuclear Magnetic Resonance (NMR)
NMR has not been widely used in the analysis of proteins and peptides due to the pure digitization of the signal, the excessive overlapping range (due to the relative molecular mass) and the weak signal. With the application of two-dimensional, three-dimensional and four-dimensional NMR, the development of molecular biology and computer processing technology has made NMR gradually become one of the main methods for the analysis of such substances. NMR can be used to determine the amino acid sequence, quantify the composition of each component in the mixture, and the like. However, there are still many problems to be solved in protein analysis, for example, how to make a protein with a large molecular weight have a specific shape to facilitate quantitative and qualitative analysis, and how to reduce the time of data processing [24]. Many scholars are conducting research on these issues. Although it is rarely used in protein analysis, NMR is very useful in analyzing small peptides containing less than 30 amino acids in the molecule, and can overcome the shortcomings in the above protein analysis to achieve rapid and accurate analysis.

2.3 In addition to the above methods, amino acid composition analysis, amino acid sequence analysis, field analytical mass spectrometry, IR, UV spectroscopy, CD, circle and chromatography, bioassay, radioisotope labeling and immunology methods have been applied to peptides The results of the substance are identified, analyzed and tested.
The above briefly introduces the common methods and the latest research directions for the separation and analysis of peptides in recent years. With the continuous development of science and technology, there will be many new methods of separation and analysis, so the research in this field has broad prospects.

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