Studies On The Dual-retention Mechanisms Of Peptides And Proteins On Liquid-solid Interface In Liquid Chromatography

The retention mechanism of peptides and proteins at the solid-liquid interface in high performance liquid chromatography（HPLC）is an advanced and active research topic in the fields of biomedical and bioengineering in recent years.Reversed-phase liquid chromatography（RPLC）has been broadly applied in proteomics,and separate and analyze the therapeutic peptides.In the analysis of peptides,the problem of overlapping and irregular peaks has not been solved yet.Traditional method of protein purification takes a long time and has a low recovery,in which the first step is precipitation and the second step is purification by liquid chromatography.In all one-dimensional and two-dimensional liquid chromatography separation,it is necessary to improve selectivity,find differences and maximize separation space.With the difference from small molecules,the retention behaviors of peptides and proteins in HPLC are very different due to their three-dimensional structure.In this paper,the steadary-migration retention behaviors and a dual-retention mechanism of different peptides,such as 6 kinds of tripeptides、2 kinds of oligopeptides and 4 kinds of polypeptide on liquid-solid interface in RPLC were studied systematically.Moreover,the retention behaviors and thermodynamic of proteins in hydrophobic interaction chromatography（HIC）were further discussed.It is very important to separate the target peptides and protein drugs on a preparative scale in industry.In this thesis,the dual-retention mechanism of peptides and proteins in chromatography was studied.The main contents are as follows.1.Literature Review.By citing 156 references,the progress in the separation of polypeptides and proteins by LC,the retention model of solutes in liquid chromatography,the relationship between retention model and retention behavior,and stoichiometric displacement theory and its application were reveiwed.2.Steady-migration retention characteristics of peptides under gradient elution in RPLCIt was firstly found that the retention characteristics of peptides under gradient elution in RPLC was dominated by two variables of the steady region（SR）and migrationregion（MR）and the steady region contributed more to the retention of solute.The relationship between the retention time and sampling time of 12 kinds of peptides in RPLC was simulated.The regression analysis method was esTablelished to predict peptide retention,and critical migration time point(T_CMP)was calculated.The stoichiometric displacement parameters of logI,Z,j and logjof peptides in RPLC were determined based on the stoichiometric displacement theories（SDT）,which were used to characterize the interaction between the peptides and the stationary phases.3.A dynamic separation method for minor-adjustments of the retention of peptides in RPLCThe minor-adjustment of the retention of peptides,induced by varying the mobile phase flow-rate（MPF-R）,is a new method to identify and improve the selectivity of overlapping peaks at the same time.The changes of peptide retention caused by different flow rates are mainly due to the bond breaking rate of the interaction between the SR domain polypeptide molecules and the stationary phase,and the energy transfer between the flow phase and the stationary phase in the MR region.The two variables also depend on the type of peptide.Satisfactory results were obtained by separating 2 kinds of oligopeptides and 4 kinds of polypeptide and the digest of lysozyme by trypsin.This method is expected to be applied to the quality control of peptide drugs,the separation of peptide peaks in peptide mapping and“bottom-up MS”in proteomics,and even peptide purification on a preparative scale in industry.4.The migration of proteins in hydrophobic interaction chromatographyIt was found that the retention of intact proteins under gradient elution in hydrophobic interaction chromatography（HIC）was also controlled by two variables,the steady region（SR）and the migration region（MR）.In the SR,the proteins were immobilized by the strong interactions with the stationary phase.In the MR,the proteins also interacted with the stationary phase,but they moved normally.Thus the retention time depended on their partition coefficients.The SR can be used as an operation space（OP）.The OP can also be employed for all assisted operations in online 2D-LC.Based on the steady region/migration region optimization strategy developed in this study,a crude extract ofa-amylase from industrial amylase sample was purified using online 2D-LC in 40 min.This approach can be used to expedite the purification of drug-target proteins and applied in the pharmaceutical industry.5.Thermodynamic behaviors of proteins in hydrophobic interaction chromatography.The thermodynamic behaviors for five proteins in hydrophobic interaction chromatography（HIC）were investigated in the temperature range from 0℃to 50℃.The thermodynamic parameters(ΔH^?、ΔS^?、ΔC_p^?、ΔG^?)of these proteins were determined by using non-linear Van’t Hoff equation.It was found that the retention of proteins in HIC was entropy driven.According to the stoichiometric displacement theory,the values of logI and Z in different temperatures were measured and the mechanism of the“steadary-migration phenomenon”from thermodynamics was explained.