Frontier Analysis Of Capillary Electrophoresis To Study The Interaction Between Cytochrome C And Drugs

Cyt c is a heme protein located in the mitochondrial membrane space.In addition to being a biological electron transporter,cyt c is also an apoptosis marker.As a specific target to regulate cell apoptosis,it can be used to develop tumor-targeted therapeutic strategies based on apoptosis.Aptamer is a kind of artificial single-stranded oligonucleotide that can specifically recognize the target.Its specific recognition ability is similar to that of antibodies,with stable properties,no immunogenicity,easy modification and immobilization,and low cost.It is widely used in detection and delivery systems for different targets.However,most of the current analytical applications surrounding Aptamer remain at the methodological research stage,especially in vivo applications are far from popular with antibodies.In order to promote the popularization and application of Aptamer,an"artificial antibody"in vivo,and guide the continuous optimization of its structure and function,it is urgent to construct a simple and efficient method for affinity and specificity analysis.Frontier analysis of capillary electrophoresis(FACE)introduces a large volume(about 100n L)of pre-equilibrated host-guest mixed sample into the capillary,and determines its binding constant and binding measurement relationship based on the platform peak of the guest molecule.This topic attempts to use the FACE method to study the interaction between the carrier cyclodextrin and the drug,the target protein cyt c and Aptamer,and further improve the detection sensitivity through the CE-MS combined technology,and use the mass resolution of MS to achieve the detection of proteins and their complexes.Effective separation is used to guide Aptamer structure and function optimization,and promote its practical application in the field of tumor targeted therapy.Based on the above ideas,the research content of this topic is as follows:1.Use the FACE method to study the interaction between the carrier?-cyclodextrin(?-CD)and two non-steroidal drugs(naproxen and diclofenac).Firstly,the injection pressure,injection time and separation voltage of CE were optimized,and the rapid analysis of FACE was realized through pressure assistance.The binding constants of naproxen and diclofenac were 587.84 L/mol and 150.69 L/mol,respectively,and both Combine in a ratio of 1:1.The CE-MS technology is further used to construct a new FACE-MS method.The system optimizes the separation voltage and auxiliary pressure at the CE end,the electrospray voltage and ion transfer tube temperature at the MS end,and the solution conditions.Among them,the sheath fluid composition(methanol/water:50/50,v/v)and flow rate(1.0?L/min)of the interface play a key role in the stability and reliability of the MS signal.The interaction between?-CD and naproxen was studied by the FACE-MS method,the peak height change trend of the naproxen plateau peak was obtained by the mass resolution of MS,and the binding constant K of the two was determined to be 203.89 L/mol,and the binding ratio It is 1:1.The results of the two methods of FACE and FACE-MS are very close,and basically consistent with those reported in the literature.2.The FACE method is used to study the interaction between the target protein cyt c and the three Aptamers(Apt 40,Apt 61 and Apt 76),by optimizing the experimental conditions(including background buffer composition,injection pressure,injection time,Separate the voltage and auxiliary pressure,etc.)to obtain a stable cyt c plateau peak.Taking into account the partial dissociation of the formed complex when migrating in the capillary,a mobility correction method is proposed here to improve the accuracy of the results,and the binding constant(Apt 40:1.09×10~5 L/mol;Apt 61:1.62×10~5 L/mol;Apt 76:3.54×10~5 L/mol),and they are combined with cyt c in a ratio of 1:1.The results are basically consistent with those reported in the literature,indicating that the FACE method has been successfully used in the analysis of the interaction between protein and Aptamer.3.First,the fluorescence titration experiment was used to obtain the binding constant of cyt c and Aptamer(Apt 40:1.51×10~5 L/mol;Apt 61:6.25×10~5 L/mol;Apt 76:8.68×10~5L/mol),which verified FACE The experimental results obtained by the method evaluated the feasibility of FACE method to study the interaction between protein and Aptamer.In order to further improve the sensitivity of detection and realize the discrimination of proteins and their complexes in different conformations from the two dimensions of platform peak intensity and m/z,the constructed FACE-MS method was extended to analyze the interaction between cyt c and Apt 40.Through careful optimization of experimental conditions and instrument parameters(especially cyt c concentration,injection time and sheath fluid composition)to obtain stable and reliable mass spectrometry signals,the effective separation of proteins and their complexes can be achieved with the help of mass resolution of mass spectrometry.The binding constant of the two(1.57×10~6 L/mol)and the binding measurement relationship(1:1).The FACE-MS method is the first to study the interaction between biological macromolecules(protein-nucleic acid),and provides new methods and strategies for research in this field.