基于超滤辅助样品制备方法的优化

摘要

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 ultrafiltration 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.%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。