Application Of One-Dimensional Gel Electrophoresis Technique(1-DE) In Serum Comparative Proteomics Research

Objectives:1. To establish a simple and sensitive method suitable for the serum comparative proteomics research specifically for proteins with low-molecule weight and low abundance.2. Using quantitative software for SDS-PAGE gel analysis to quantify the density of electrophoresis bands, converting them into digital form for statistical analysis.3. Using bioinformatics analysis to verify the feasibility of applying this data converting method in one-dimensional gel electrophoresis (1-DE) of serum comparative proteomics research.Methods:1. Remove high aboundance serum proteins by salting out, organic solvent, and affinity chromatography. Compare these mathods in regard to the effects of high abundance protein removal and low-molecule weight protein retention by protein concentration determination and SDS-PAGE electrophoresis analysis.2. Separate the samples by the second floor of 15%Tris-SDS-PAGE, three floor of 15%Tris-SDS-PAGE, Tricine-SDS-PAGE, urea Tricine-SDS-PAGE respectively, compare the various methods taking effect on low-molecule weight protein separation.3. Use a quantitative gel imaging software Quantity One to quantify each band in the electrophoresis map and then convert the graphical results into digital results.4. Those techniques for normal people and liver cancer serum protein analysis verify these technologies, and compare to the results that one-dimensional gel electrophoresis techniques made in our study,establish the feasibility of our study.Results:1. Acetonitrile precipitation could remove about 90% of high abundance proteins in serum, while more low molecular weight protein (below 15kD) remained; salting out precipitation and affinity chromatography could remove most of the high abundance proteins in serum, yet these two methods retended less low molecular weight proteins. Affinity chromatography column method could specifically remove albumin and immuno globulin .2. Urea-Tricine-SDS-PAGE could satisfactorily separate low molecules weight proteins in serum, Protein bands below 15kD were clearly visible. Its separation efficiency was better than that of the second floor of 15% Tris-SDS- PAGE,three floor of 15%Tris-SDS-PAGE and Tricine-SDS-PAGE. Therefore, it is more suitable for the following analysis of low molecular weight bands by the Quantity One software.3. After analysis of gel images with Quantity One the gel pictures were converted to digital data. Statistical analysis (SPSS 13.0) showed thatρ<0.05 , as amended,there are 10 bands with significant difference.Conclusions: 1. Methods for serum sample preparation were established and optimized. The acetonitrile precipitation method had advantage over other methods in that it could remove the vast majority of high abundance proteins, while keep more low molecular weight proteins. This method is suitable for separating serum proteins below 15kD.2. Methods for differential SDS-PAGE electrophoresis were established and optimized for serum samples. Urea-Tricine-SDS-PAGE electrophoresis could clearly separate seven bands below 15kD, This method has an obvious advantage in separating low molecular protein below 15kD.3. To analyze one-dimensional gel electrophoresis map, the results need to be treated by the gel quantification software Quantity One. The method is feasible for the study.4. After analyzing serum proteins from normal people and liver cancer patients with the above methods, we have proved that one-dimensional gel electrophoresis technique is feasible in serum comparative proteomics research.