[采用改良三氯乙酸/丙酮沉淀法去除肥胖者血浆中的高丰度蛋白质]

[Removal of high-abundance proteins in plasma of the obese by improved TCA/acetone precipitation method].

作者信息

Wang Jun, Feng Liru, Yu Wei, Xu Jian, Yang Hui, Liu Xiaoli

机构信息

Shenzhen Center for Chronic Disease Control, Shenzhen 518020, China.

出版信息

Wei Sheng Yan Jiu. 2013 Sep;42(5):741-7.

PMID:24218878

Abstract

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/acetone 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.

摘要

目的

开发一种改进的三氯乙酸（TCA）/丙酮沉淀法，用于去除肥胖者血浆中的高丰度蛋白质。

方法

研究了TCA/丙酮溶液体积（样品的1、3、4、5、6、8、10和20倍）和TCA浓度（10%、30%、50%、60%、70% TCA/丙酮溶液），以优化样品制备条件。采用SDS-PAGE分离并检测上清液和沉淀物中的蛋白质。首先通过SDS-PAGE确定TCA/丙酮溶液的最佳浓度。通过二维SDS-PAGE验证10% TCA/丙酮溶液处理沉淀中的蛋白质和新确定浓度的TCA/丙酮溶液处理沉淀中的蛋白质。然后通过纳米HPLC-Chip-MS/MS分析沉淀和上清液中蛋白质经胰蛋白酶消化后的产物，以验证处理血浆的最佳浓度。

结果

TCA/丙酮的最佳体积为样品的4倍，TCA/丙酮过多或过少都会降低高丰度蛋白质的去除效率。丙酮溶液中TCA的浓度应为60%，与10%、30%、50% TCA丙酮溶液相比，其去除血浆中高丰度蛋白质的效果可能更好。如果TCA浓度超过60%，由于蛋白质快速沉淀，重现性会差得多。质谱鉴定结果表明，用60% TCA/丙酮（4倍样品体积）处理的人血浆比10% TCA/丙酮处理的血浆能验证出更多低丰度蛋白质。