Screening And Verification Of New Protein Markers In Maternal Serum For Early Diagnosis Of Down's Syndrome

Objective:The project utilizes comparative proteomics to analyze and validate the differential expression proteins between Down Syndrome (trisomy 21) and normal fetus mid-gestation in matrix serum by two-dimension electrophoresis and mass-spectrometric detection, and to sieve the specific new marker of matrix serum proteins associated with DS, it may lay the foundation for the Down Syndrome fetus'early non-trauma prenatal screening.Methods:1. Extraction of the serum proteinsAfter centrifugating the serum samples, imbibe the interlayer, remove the greases of supernatant. Filter it with filter membrane after diluting, then remove high abundant proteins with Agilent Multiple Affinity Removal Column. Utilize 5K hyperfiltration tube to condense and demineralization. Finally, quantitate the proteins with Bio-Rad protein assay reagent, Cryopreservation standby.2. Two-dimensional gel electrophoresisManipulate procedure accords to the User Guide of isoelectric focusing system of Gorgetc. And IPGphor etc. methods. Repeat the experiment 3 times on every sample, proceed isoelectric focusing and SDS-PAGE two-dimensional gel electrophoresis after sampling, then silver staining, subsequently, dry the gel with gel dryer(Bio-Rad company), to obtain electrophoretic film.3. Establishment of the spectrum of differential proteinsCapture the electrophoretic image of serum proteins with Image scanner and Lab scan soft from The electrophoretic gel by staining and drid. Then utilize Imagemaster 2D Elite4.01 analyzing soft to two-dimensional gel electrophoresis silver staining of experimental group and normal one at intensity detecting, background pruning, matching, correction of 1D and 2D,establishing equilibrate gel, quantization and capturing the gray scale information of proteins fleck one by one, established the spectrum of differential proteins.4. Biological mass-spectrometric analysis of differential protein pointsSelect differential protein flecks coming from image analysis, get the flecks from the gel to decoloration,dehydration,reduction,enzymolysis and extraction. Homogenize the samples and ground substance solution fully, apply the mixed liquor to stainless steel templet, then air drying. Put the templet into velocitron to analyze and to capture the peptide mass'finger print map.5. Data base querying of differential protein spotTo search the peptide mass fingerprint in NCBI. Query parameters: choose human as species classification.; enzyme selected Trypsin;choose 1 as permitted restriction sites ; set three possible modification, carbamidomethyl (C), oxidation (M) and pyro - glu (N-temE ),and amount of error is 0.2 Da ;choose 100 ppm as peptide fragment tolerance; ion choose MH+ and monoiso-tope;input Mascot Search,then you can rearch database.6. Validation with western blottingValidate the differential proteins sieved from the immune blotting methods of SDS-polyacrylamide gel electrophoresis, transmembrane, hybridization etc, obtain autoradiography x-ray film; the film is scanned by visible light/ultraviolet gel scanning analyze system(UVP, American), then obtain integral optical density table with analytic soft system(LabworksTM Analysis Software,American)Results:1. Two-dimensional gel electrophoresis detectionIn this study, there are four cases of normal groups and four cases of test groups which are Down's syndrome, and each of maternal serum had a 2-DE. Protein spots were clearly discernible. The number of protein spots was about about 612 to 754 and 648 to 731. Image shows that in test groups and in normal groups, protein spots were mainly located at the molecular weight of 15.5 ~ 120kD, pI 4.0 ~ 8.0, especially at the 35 ~ 85kD, pI 4.5 ~7.5 region. Distribution of dense protein spots illustrates that extremely acidic and alkaline protein are less in serum. The two gel maps were analyzed with imagemaster 2D Elite 4.01 software.The number of detected protein spots was average (684.3±71.0) in normal groups, and (690.0±41.5) in test groups. Image synthesis was taked in the two groups whose spots were clear, and then two synthetic gels were generated and compared with each other groups. Basing on the software analysis and visual observation, there are 19 protein spots test groups specific expressed or significant increased in test groups, there are 10 decreased. It is said that there are 29 protein spots expressed significantly different in Down's syndrome compared with health adult.2. Mass spectrometric detection8 protein spots which were selected in 29 significantly different protein spots were taked mass spectrometric detection. It is found that 8 proteins in database with the possibility of matching have statistical significance. These differential proteins are GTPase, Factor B Serine Protease Domain, Beta2-Glycoprotein I, Complement factor H-related protein 1 precursor, Kininogen 1 isoform 2, Crig Bound To C3c, Human Complement Component C3c,Crystal Structure of Lipid-Free Human Apolipoprotein A-I.respectively .3. Immunoblotting detectionWestern blot analysis was used to detect the expression of dGTPase and Beta2-Glycoprotein I within 8 protein spots. It's results show that the straps of DS pregnancy serum specimen were brighter than normal pregnancy serum specimen, it increased the degree of confidence of proteins. Densitometric analyses were performed using LabworksTM Analysis Software. The results show that the relative amount of proteins in DS pregnancy serum specimen were higher than normal pregnancy serum specimen above 2-fold. It proves furtherly that the findings of proteomics are correct.Conclusions1,To establish different protein patterns of DS and normal maternal serum with 2-DE technique, and filter out 29 significant differently expressed protein spots ,among which there are 19 protein spots expressed significant or increased in maternal serum ,10 decreased. 2,To establish the peptide fingerprinting of different protein with biological mass spectrometry analysis ,and identify 8 DS-related maternal serum markers.3,To validate dGTP enzyme and Beta2-Glycoprotein I with WB , and the results of them were significantly higher than in the normal maternal serum, which was coincided with the mass spectrometry results.It is suggested that dGTPase and Beta2-Glycoprotein I are two new markers of DS maternal serum .It may be used as early screening of DS.