儿童原发性肾病综合征尿蛋白质组样品预处理方法的研究

Pretreatment methods of urine proteomics in children with primary nephrotic syndrome

目的优化儿童原发性肾病综合征尿蛋白质组样品的预处理方法。方法取原发性肾病综合征儿童尿液,采用称量、SDS-PAGE及双向电泳(2-DE)等方法比较不同pH、冷丙酮沉淀不同时间等条件下所得到的蛋白质,优化蛋白提取的条件。结果 pH 2.7条件下所得到的蛋白种类最多;冷丙酮沉淀0.5 h获得的蛋白量明显少于1 h及2 h的蛋白量(P<0.05),而冷丙酮沉淀1 h和2 h获得的蛋白量没有显著性差异。pH 2.7条件下冷丙酮沉淀1 h为样品预处理的最佳条件,可获得满意的2-DE分离效果。结论 pH 2.7条件下,冷丙酮沉淀1 h为尿蛋白质组样品预处理的最佳条件。

Objective To optimize a pretreatment method of urine proteomics in children with primary nephrotic syndrome.Methods Urine from children with primary nephrotic syndrome was treated in different pH and isolated by cold acetone precipitation for different durations.Then the amounts and kinds of proteins were compared by quantify,SDS-polyacrylamide gel electrophoresis（SDS-PAGE） and two-dimensional electrophoresis（2-DE） in order to optimize a way to deal with urine protein.Results Most proteins were obtained at pH 2.7.The amounts of protein precipitated by acetone for 0.5 hr was obviously less than those precipitated for 1 and 2 hrs（P0.05）,while there was no significant difference between the amount of protein precipitated for 1 and for 2 hrs.Protein precipitated by cold acetone for 1 hr at pH 2.7 was selected as the best pretreatment method.Satisfactory 2-DE maps can be acquired.Conclusions Urine protein can be best obtained at pH 2.7 and precipitated by cold acetone for 1 hr.