Research In Separation,Purification And Quantification Of Important Protein Biomarkers

Compared to the pure reference material,the matrix reference material is similar to the real condition of the test substance,and the quantitative measurement result is more accurate.This experiment studied the purification and quantification of biomarkers in serum.Based on the immunomagnetic beads enrichment process,the separation and purification of low-abundance biomarkers in serum was achieved,and a method for accurately quantifying the target was established.It is a serum matrix reference material.Development provides a reference.Due to the large variety of proteins in the serum matrix,in order to avoid the matrix effect,to achieve the separation and purification of specific low-abundance proteins to reach the detection limit of the quantitative method,specific purification of the target substance is required.In this study,immunomagnetic beads enrichment technology was used to purify target proteins in serum by binding magnetic beads of monoclonal antibodies to enrich target proteins.The purified protein was decomposed into characteristic peptides by enzyme digestion,and the purpose of protein quantification in serum was achieved by quantifying the characteristic peptides.In order to accurately determine the purity of synthetic peptides that serve as the basis for the quantification of peptides,this study used synthetic glucagon as an example to establish a quantitative method for the determination of the purity of synthetic peptides in solid phase.The impurities in the target were accurately quantified,and the total mass minus the impurity mass yielded a purity of 89.64%.This result is more accurate than the commonly used HPLC method(98.37%).The method was used to separate,purify and quantify the small molecule polypeptides in the serum.In this study,the diabetes biomarker C peptide was selected as an example.Separation and purification of C-peptide in serum was achieved by immunomagnetic bead enrichment technology,and a method for the quantification of C-peptide in serum by isotope dilution mass spectrometry was established.By adding the isotope-labeled C peptide as a reference,the enrichment process of the immunomagnetic beads was optimized,and the enrichment efficiency of the immunomagnetic beads was 80.22%.The isotope dilution mass spectrometry method was used to quantify the isolated and purified C peptide in the serum,and the concentration thereof was 10.80 ng/mL.Through the optimization of the binding efficiency of magnetic beads and antibodies,the binding efficiency of antibodies and proteins,the loading of antibodies,and the elution time factors in the process of enrichment of magnetic beads,The process of separating and purifying C-reactive protein in serum by polystyrene beads combined with monoclonal antibodies was designed.The process of separating and purifying C-reactive protein in serum.The C-reactive protein was digested by trypsin and the efficiency of enzymatic cleavage was increased by adding ultrafiltration technology.The coverage of peptide maps increased to 86.16%,and its characteristic peptide was ESDTSYVSLK(EK).By quantifying the mass spectrum of the synthetic peptide EK,the EK concentration after C-reactive protease cleavage in the sample serum was 0.10 ug/mL.Based on the relationship between the characteristic peptide and the amount of C-reactive protein material,the C-reactive protein in the serum of the sample was calculated.The content is 2.06 ug/mL.The separation,purification and quantification of serum biomarkers in this experiment provide reference for the development of serum matrix reference materials.