Quantification Of D-dimer In Human Plasma Based On Isotope Dilution Mass Spectrometry

Protein biomarkers are important indicators for disease diagnosis.Their concentration in human body are closely related to disease process,and they can be used as effective bases for disease diagnosis and treatments.In clinical tests,accurate and reliable determination results must be traceable and comparable,so it is necessary to establish corresponding reference materials and reference methods.Proteins have the characteristics of large molecular weight,complex structure and instability.There are many influencing factors in quantitative determination,and the coefficient of variation of test results is high.The reliability and traceability of the test results of such markers are still at a low level.Therefore,the development of accurate quantitative methods for protein biomarkers is of great significance for the standardization of their measurement.D-dimer is the smallest product of fibrin degradation in the human body and is an important biological indicator for clinical exclusion of deep venous thrombosis(DVT),disseminated intravascular coagulation(DIC)and pulmonary embolism(PE).At present,monoclonal antibody-based methods are mainly used for quantitative detection of D-dimer in clinical practice,including latex agglutination,enzyme-linked immunosorbent assay or immunofluorescence assay(ELISA/ELFA),whole blood agglutination and colloidal gold immunofiltration.In fact,in addition to the smallest D-dimer,clinically detected D-dimer antigens also contain fibrin degradation products(FDPs)with multiple D-dimer characteristic structures.Because the measurement of D-dimer is not clear,there is no corresponding standard material and reference measurement method in the world.Moreover,due to the difference in binding ability between different monoclonal antibodies and D-dimer antigens with different structures,the results of various detection methods are poor in comparability and low in accuracy,which greatly limits the application of D-dimer in clinical diagnosis and treatment.Isotope dilution mass spectrometry is considered to be the benchmark method for the development of protein quantitative reference program,which has the advantages of high accuracy,high sensitivity,traceability and wide detection range.However,there is no report on the high accuracy determination of D-dimer based on this method.In this study,based on protein digestion and isotope dilution mass spectrometry,a characteristic cross-linked peptide in D-dimer trypsin digest production were used to replace D-dimer,and an accurate quantitative method of D-dimer based on isotope dilution mass spectrometry was established.The main research contents are as follows :(1)By optimizing trypsin digest conditions of D-dimer protein and the conditions of chromatography and mass spectrometry in quantitative detection,combined with isotope dilution mass spectrometry,a signature peptide-isotope dilution mass spectrometry method was established to quantify the concentration of D-dimer in solution.This method has a good linearity in the mass range of 0.05-5 μg,and the linear correlation coefficient is 0.99.(2)Based on immunomagnetic separation technology,D-dimer in human plasma was separated and extracted,and the enrichment and elution conditions were optimized.D-dimer plasma samples with different concentration levels were prepared by adding method.The plasma samples were separated and extracted according to the optimal conditions.The concentration of D-dimer in plasma samples was successfully quantified by signature peptide-isotope dilution mass spectrometry method.The recovery rate of the method was between 76.68% and 119.32%,the quantitative limit was 0.42 μg/m L(DDU),the linear range was 0.42-17.01 μg/m L(DDU),and the linear correlation coefficient was 0.99.(3)The results of the signature peptide-isotope dilution mass spectrometry method and the commercial immunoturbidimetry kit in plasma were compared.Since the measurement was determined,the sensitivity and accuracy of the signature peptide-isotope dilution mass spectrometry method were better.The quantitative results had a good correlation with the results of the kit,and the Pearson coefficient was 0.99.In this study,a signature peptide-isotope dilution mass spectrometry method for quantitative D-dimer was successfully established,and the Ddimer in solution and plasma was accurately determined.This method clarifies the measurement of D-dimer quantification,improves the accuracy and comparability of D-dimer quantitative detection results,and has a good correlation with existing clinical quantitative methods,laying a foundation for the development of D-dimer reference materials and reference methods.