The Validation Research Of CHD Biomarker And The Establishment Of New MRM Absolute Quantitative Method

Objective:To validate CHD biomarker of the differences proteins found on the early stage research by isotope labeling MRM absolute quantitative method,and assess the screening efficiency.To establish a kind of label-free MRM absolute quantitative method.Method:Appling statistics and bioinformatics technology to screen 329 kinds of different proteins which were found by iTRAQ technology and MALDI-TOF-MS/MS technique on our early stage research.Those serum proteomic expression profiles were taken the relative quantitative analysis between the VSD patients,ASD patients,TOF patients and normal control group.The peptide fragment and ion pair of the validated protein were choose with the help of UniProt database and Skyline software.The MRM mass spectrum scanning method was optimized.For the choose of sample pretreatment method,we compared three methods including the acetonitrile precipitation,acetone precipitation and MARS Hu-7 he Affinity column.We compared of BCA protein quantitative method and Bradford protein quantitative method for the determination of total protein.We also examines the effect of urea and RapiGest as denaturant to digest protein.According to the optimal pretreatment methods,20 cases of VSD patients and 20 cases of normal control children serum were deal with.The content of 5 kinds of proteins including ICAM-1/CD54,VE-cad,IGF-2,TSP-1 and Apo-E were detect by isotope labeling combination of MRM absolute quantitative methods and ELISA methods.The quantitative results of two methods were evaluated the screening index.In order to reduce the cost of protein detection,a kind of protein absolute quantitative method about label-free peptides standard addition method incorporation with MRM was established.We comparied with the stable isotope labeling method in the precision of the method,recovery rate and quantitative determination of repeatability and linear range.Result:1.Selection of unproven.protein:the protein ratio is transformed the protein abundance by log2.For the standard deviation with all the protein ratio is0.799,so the threshold is 0.799.If the protein ratio is above 0.799 or below-0.799,it is significantly different protein.According with the threshold,there are 14 protein is raised and 8 protein is reduce in VSD group.There are 10 protein is raised and 16 protein is reduce in ASD group.There are 13 protein is raised and 9 protein is reduce in TOF group.Those protein also analysis by enrichment of Gene Ontology.Based on the two factors of genes analysis and proteins ratio,we choice of those protein which the protein ratio was greater than 0.799,or less than-0.799,and involved in biological processes,molecular function and cellular component.Finally we chose 14 protein from 329 protein as for validation protein.2.We preferred download in all validation protein sequences in UniProt database,then imported the protein sequences into the SKYLINE software and established the initially MRM mass spectrometry method of 14 kind of protein,136 peptides and 544 pair of ion pair.In the optimization peptides stage,through actual testing serum samples,only 37 peptides arid 278 pair of ion pair of the 14 protein are qualified for optimization of ion.In phase ion pair optimization,some ion pair which have the poor peak shape or peak strength is low,or the peak width is too large ion pair were weeded out.Finally we select 42 ion pairs in 14 peptides of nine proteins.On the basis of the selected peptides and ion pair,collision energy and cluster voltage of each peptides and each ion pairs were optimized separately.The peak shape and peak strength is greatly improved compared to that before optimization.3.The pretreatment method of sample selection:On the choice of protein accumulation method,we compared three methods include the acetonitrile precipitation,acetone precipitation and Multi Affinity Removal Spin Cartridge MARS Hu-7 he Affinity column.MARS Hu-7 he affinity column effect is about 5 times of acetone precipitation,10 times of precipitation acetonitrile,so we chose the MARS Hu-7 he affinity column.protein concentration measurement methods.On the choice,of total protein quantitative method,We examine the BCA protein quantitative method and Bradford.The experimental results show that the two methods determination of protein content is almost similar,so we choose the BCA method.About protein digestion method selection,we examine two kinds of mode which the urea and RapiGest TM SF as denaturant of enzyme.The experimental results showed that if urea as denaturant,protein enzyme content of is serum samples relatively high and the effect of digestion is more completely.4.The absolute quantitative of 5 kinds of proteins including ICAM-1/CD54,VE-cad,IGF-2,TSP-1 and Apo-E were detect in the serum of 20 cases VSD group and 20 cases of normal control group by isotope labeling combination of MRM absolute quantitative methods and ELISA methods.The content of VE-cad,TSP-1 and Apo-E were significant difference in the two group.It means ICAM-1/CD54 and IGF-2 is false positive.For determining VE-cad and Apo-E,MRM and ELISA method are similar on the ROC curve,sensitivity,specific,false negative rate and false positive rate,Youden’s index,likelihood ratio,coincidence rate,Kappa value and predictive value.For determining TSP-1,ELISA is superior to MRM.Compared with ultrasonic cardiogram methods in screening for CHD patients,two methods of determination of VE-cad,TSP-1,Apo-E three protein sensitivity and supersonic and enchanted the graph method,the sensitivity of similar,but the specificity is lower than the specificity of the echocardiography.5.A kind of protein absolute quantitative method about label-free peptides standard addition incorporation with MRM was compared with the stable isotope labeling method.The experimental results showed that the precision,recovery rate,quantitative determination of repeatability and linear range is similar of the two methods.Applying these two methods to determine 25 cases of congenital heart disease in patients with ventricular septal defect.We found that the determination of paired t test of Apo-E in serum enzyme was no statistical significance.The equivalence test results show that two methods are equivalent.It suggests that establish the label-free peptides standard addition method can take the place of the stable isotope labeling method and could be used to validate the other biomarker proteins in congenital heart disease.At the same time because of the label-free peptides standard addition method does not need to syntheses of expensive absolute isotope labeling peptides,so this method have the advantages of economy,simple,high accuracy,and good stability.Conclution:1.VE-cad,TSP-1,Apo-E protein are biomarkers in CHD patients serum;2.MRM and ELISA determination of VE-cad,TSP-1,Apo-E protein can be used as a screening test for CHD.3.The label-free peptides method may can replace the stable isotope labeling method.As a new simple economic method,it has higher application value.