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Development Of Fractionation And Identification Methods For Complex Protein Systems And Biomarkers Discovering In Diabetes-related Serum

Posted on:2008-06-05Degree:DoctorType:Dissertation
Country:ChinaCandidate:R X LiFull Text:PDF
GTID:1104360215955091Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Proteins from biological samples, e.g. plasma/serum, are usually very complex and have broad dynamic-range. Moreover, disease-related biomarker proteins are often present in serum with low abundance. Therefore, developments of efficient, sensitive, and high-throughput protein fractionation and identification methods are necessary for successful biomarker discovery. In this study, we developed and evaluated four different protein fractionation and identification strategies. Subsequently, we used these methods, in conjunction with bioinfomatic tools, to study both animal and human diabetic models, and found several potential plasma/serum protein markers for diabetics.We could identify thousands of different proteins from plasma sample of around 10 mg total protein by the combination of liquid focusing and multidimensional protein identification technology, as well as the combination of MDLC-2D-LC-MS/MS. Therefore, both of these two methods are compatible for proteomic studies of plasma and other complex samples.Using LC-MS/MS, in conjunction with SDS-PAGE, we identified a total of 13,206 serum proteins from type-2 diabetic model-GK rats and control Wistar rats. After removing developmental influence using subtractive proteomics, we found 283 differentially-expressed proteins, and these proteins are likely related to the pathogenesis of diabetes. Furthermore, we compared the protein profiles of diabetic and non-diabetic serum from human subjects (n=5 for each group). We found that many proteins involved in complement and coagulation cascades are significant up-regulated in diabetic serum. The up-regulation of several such proteins (C3, C3a, C3b and C5b-9) was confirmed by Western blot. Therefore, we conclude that the complement activation is present in the diabetic serum, and it might be involved in the pathogenesis of diabetes.In order to simplify the experimental procedure and make it suitable for high-throughput analysis, we replaced SDS-PAGE with ethanol precipitation for sample preparation prior to LC-MS/MS. Using this strategy, we analyzed 10 non-diabetic and 17 diabetic serum samples, and found 241 differentially-expressed proteins. Further analysis found that 6 proteins had strong correlation with FPG (fasting plasma glucose) and PG2H (2-hours plasma glucose).In summary, we developed four fractionation and identification strategies to study low abundant proteins in plasma/serum. Using these methods, we found several potential plasma/serum biomarkers for diabetes mellitus. Our data provide valuable insights to future pathophysiologics and diagnostics studies on diabetes mellitus. Meantime, our proteomics strategies can also be used to find plasma/serum biomarkers related to other diseases.
Keywords/Search Tags:Complex protein system, liquid isoelectric focusing, label free quantitative strategy, complement and Coagulation cascades, diabetic associated biomarkers
PDF Full Text Request
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