Difference Analysis Of The Amniotic Fluid And Plasma Proteomics

Amniotic fluid embolism is a serious complication, which the amniotic fluid enter into the blood circulation of mother during delivery date. The reports about the incidence is inconsistent, which is 1: 5000-1:80000. The mortality rate is as high as 70-80% at the full-term pregnancy, the complicated clinical manifestations are the mainstay of diagnosis. Now we still lack a received standard of the early detection for various pregnancy related diseases,such as amniotic fluid embolization, and many biomarkers being used are not fully individual detection rate and specificity, so we need to develop various biological markers, and look for more specific markers further.The comparison in which the protein samples are from different tissues or different physiological conditions, is one possibility for which is used for the identification of biomarkers of a disease. Currently proteomics analysis has been widely used in the study of body fluid. Many research groups use different methods and different proteins database, and the strict level of protein identification is also different. For that, it is difficult for us to evaluate the accuracy of each protein database, but the obtained results by virtue of the different technical routes can be complementary with each other.Objective:To compare the normal plasma and amniotic fluid proteomics by the proteomics techniques such as carrier ampholyte 2-DE and MALDI-TOF, and to find many protein markers of specific expression of in amniotic fluid, finally make the early diagnosis for various pregnancy related diseases. As it were, the expression of certain proteins in the amniotic fluid is very meaningful, which can reflect the physiological and pathological conditions in the amniotic fluid, so the proteomics analysis of amniotic fluid is a ideal step for the explanation of the pathological changes of the body in the gestation period, which will also help us find some early diagnostic markers of certain pregnancy related disease.Methods:1. Sample preparation:collect the amniotic fluid of twelve healthy women at 16-18 weeks of gestation by the amniocentesis, and the plasma of twelve healthy persons by the cubital venipuncture, then centrifuge, take out supernatant and freeze drying for preservation. 2.Sample processing:Based on the affinity chromatography method that Cibacron blue packing adsorbs the albumin, we remove the albumin that is the highest abundant protein.3.2-DE:we can observe the expression of the proteins between amniotic fluid and plasma by use of carrier ampholyte 2-DE (ISO-2DE) and silver stain technique that is compatible with mass spectrometry.4.The analysis of the gel image:first, we use the Canon digital cameras 5.0 to capture images about ISO-2DE gel,then finish the differential analysis by the PDQquest 8.0 analysis software, finally carry on detecting and matching the protein spots,and find the diffenrential proteins in amniotic fluid.5.The extraction of the hydrolized peptides in the Silver stain gel:dig out specific protein spots,then make the enzymatic hydrolysis and extract the peptides.6.Pep-tide fingerprint analysis:according to the extracted peptides, we can search and identify the possible proteins by the moverz, peakErazor and mascot software.7.Determine the differential proteins:we need to deter-mine these differential proteins in amniotic fluid by Western Blotting technology,and verify whether these differential proteins only exists in amniotic fluid, but not in plasma.8.Clone and express the gene,and prepare antibody.9.Detect the antibody titer by ELISA method.Results:1.Based on the affinity chromatography method that Cibacron blue packing adsorbs the albumin, we can see that the most albumin(Mw 66ku) has been removed from the SDS-PAGE, and the protein bands is more clear than before removing albumin. 2.After separating the proteins in plasma and amniotic fluid by use of the carrier ampholyte 2-DE techique,the results showed that there are separately about 102 protein spots and 147 protein spots in amniotic fluid and plasma.3.We find four differential protein spots by use of the PDQquest 8.0 software.4.By use of moverz, peakErazor and mascot software,we can see that there are four protein spots in the amniotic fluid, which is respectively ZAP-70, T-Box 3, IKK-βand KIAA 1145 from the peptide fingerprint analysis results.5.By use of comassie brilliant blue colorimetric method and Western Blotting technique,we can verify these differential proteins. In our experim-ent,we only analysed IKK-βand T-Box 3, the results are that they also exist in the plasma.The content of IKK-βin the plasma is,not low, and there is no obvious difference,but the content of T-Box 3 in the amniotic fluid is higher than in the plasma,which can makes some sense.Conclusions:In our laboratory,we need to remove the albumin in the plasma and amniotic fluid through Cibacron blue F3GA at first,then to analyze and compare the normal plasma and amniotic fluid proteomics by 2-DE, PDQUEST software analysis,MALDI-TOF techniques and so on,finally to find many protein markers of specific expression of in amniotic fluid.This will provide the research foundation for the early diagnosis of various pregnancy related diseases,such as amniotic fluid embolization, and may be a supplement for the results that obtained by the other technical routes.The direction for future study:We continue to verify the differential proteins between amniotic fluid and plasma by the Western Blotting technology.If the differential proteins are confirmed, we can clone and express the genes, prepare the antibody, and detect the antibody titer by ELISA method.