基于超滤辅助样品制备方法的优化简

目的：基于超滤辅助样品制备（FASP）方法的出现使得使用去污剂（如SDS）的蛋白质提取方法与溶液内酶切方法得以兼容，因此提高了难溶性蛋白的鉴定数量。然而，超滤膜的非特异性吸附作用依然会造成蛋白的损失。我们拟针对该方法存在的问题对其进行改进。方法：对FASP方法进行了蛋白酶切条件、洗脱液选择、洗脱次数的改进；为测试优化方法的有效性和适用范围，选择标准蛋白BSA、鼠肝和鼠脑等3种样品进行考察。结果：相比报道的FASP方法，采用改进后的FASP方法使BSA的回收率提高了20％；经高精度质谱检测，对鼠肝、鼠脑的蛋白质鉴定结果分别比采用未优化的FASP多鉴定到2086和3592条特异肽段。结论：通过对FASP方法的优化，蛋白鉴定数量得到较大提高，该方法为蛋白质组深度覆盖研究提供了可靠的技术手段。

Optimization of Filter-Aided Sample Preparation

Objective： The filter-aided sample preparation（FASP） method makes detergent（such as SDS） to be compatible with ＂in-solution digestion＂. Therefore, the number of identified insoluble proteins was increased by LC MS/MS analysis. However, non-specific adsorption of ultrafihration device still causes sample losses. In this paper, we proposed an improved FASP method to solve this problem. Methods： The method made improvement in three aspects： protein digestive environment, eluent choice, and eluent times. We used three samples, bovine serum albu min（BSA）, mouse liver and mouse brain, to optimize the method. Results： Compared to the original FASP meth od, the recovery of peptides from BSA increased by 20% with the optimized FASP. Furthermore, 2086 and 3592 additional peptides were identified in mouse liver and brain proteins receptively with the optimized FASP method. Conclusion： By using the optimized FASP method, we improved the level of protein identification, thus provided a reliable technique for improving the coverage of proteomics study.