摘要
目的对传统的三氯乙酸(TCA)/丙酮沉淀法进行改良,探索一种较好的高脂血浆的高丰度蛋白去除方法。方法分别用不同体积(1、3、4、5、6、8、10和20倍样品体积)、不同TCA浓度(10%、30%、40%、50%、60%、70%)的TCA/丙酮溶液处理高脂血浆处理所得上清和沉淀蛋白样品分别进行一维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)验证比较,确定去除效果最佳的TCA/丙酮溶液,用该浓度TCA/丙酮溶液与含有10%三氯乙酸的TCA/丙酮溶液同时处理血浆,对所得沉淀蛋白进行二维SDS-PAGE验证,并对所得上清和沉淀蛋白质进行Nano HPLC-Chip-MS/MS鉴定比较。结果 4倍样品体积的TCA/丙酮溶液去除高丰度蛋白的效果较好,增加或减少TCA/丙酮溶液的体积都会影响去除效率;60%TCA/丙酮溶液去除血浆中高丰度蛋白质的效率比10%、20%、30%、50%TCA/丙酮好,而70%TCA/丙酮溶液处理血浆的重复性较差。质谱鉴定结果显示,4倍样品体积的60%TCA/丙酮溶液与10%TCA/丙酮溶液处理后的血浆样品相比,可鉴定更多的低丰度蛋白质。结论用4倍样品体积的60%TCA/丙酮溶液去除血浆中高丰度蛋白质的效果最为理想,尤其适用于较高脂含量的样品的处理。
Objective To develop an improved trichloroacetic acid（TCA） /acetone precipitation method for removal of high-abundance proteins in plasma of the obese. Methods Volumes of TCA/acetone solution （ 1,3,4,5,6,8,10 and 20 times of the sample） and concentrations of TCA（ 10% ,30% ,50% ,60% ,70% TCA/acetone solution） have been investigated to optimize the conditions of sample preparation. SDS-PAGE were used to separate and tested proteins in the supernatant and sediment. The best concentration of the TCA/aeetone solution was first determined by SDS-PAGE. The protein in precipitation from 10% TCA/acetone solution processing and the new determined concentration TCA/acetone solution processing were verified by 2-D-SDS-PAGE. And then the digested products of the protein in precipitation and supernatant by trypsin were analyzed by nano HPLC-Chip-MS/MS to verify which is the best concentration to process the plasma. Results The best volume of TCA/acetone is four times to sample, which less or more TCA/acetone would reduce the removal efficiency of high-abundance proteins. The concentration of TCA in acetone solution should be 60% , which may remove more high-abundance proteins in plasma than 10% ,30% ,50% TCA in acetone solution. If the TCA concentration is more than 60% , the reproducibility will be much poorer due to fast precipitation of proteins. The results of mass identification showed that human plasma prepared with 60% TCA/acetone （4 times sample volume） could be verified more low-abundance proteins than 10%. Conclusion The most desirable conditions for removal of high-abundance proteins in plasma is 60% TCA/acetone （4 times sample volume）, especially for the plasma of obesity.
出处
《卫生研究》
CAS
CSCD
北大核心
2013年第5期741-747,共7页
Journal of Hygiene Research
基金
国家自然科学基金(No.81172668
81202207)
关键词
肥胖人群
血浆
高丰度蛋白质
TCA
丙酮沉淀法
obesity, plasma, precipitation method high-abundance proteins, TCA/acetone
