Université du Québec à Montréal, Chemistry Department/Pharmaqam, Montréal, QC, Canada.
J Proteomics. 2013 Apr 26;82:166-78. doi: 10.1016/j.jprot.2013.02.001. Epub 2013 Feb 20.
A systematic approach was developed to optimize the analysis of rat liver microsomes combining ion exchange fractionation with reverse-phase chromatography coupled to high resolution quadrupole-time-of-flight mass spectrometry. A comparison was performed with several conditions to select the most efficient solubilization and proteolysis protocol to achieve highest proteome coverage. Optimal trypsin digestion conditions were achieved with SDS and heat to increase solubilization of microsomal samples, with an increase from 621 to 686 identified proteins when SDS and heat were applied. Pepsin digestion yielded complementary results, especially in terms of hydrophobic environments, thus allowing sequence coverage to be increased substantially. Several dual digestion strategies were tested, with trypsin and pepsin combined in series or in parallel. A parallel tryptic-peptic dual digestion, combining mass spectral data of single enzyme digestions, yielded the best results in terms of number of identified proteins, increasing by 29% from the best single enzyme procedure, and sequence coverage improved by 5% on average for all proteins identified. Using our complete set of data, a total of 1095 proteins were identified with less than 1% FDR, out of which 213 proteins (19.5%) were integral membrane proteins. Proteomics data have been deposited to the ProteomeXchange Consortium with dataset identifier PXD000128.
A systematic approach to optimize the proteomic analysis of rat liver microsomes by 2D-LC-MS/MS using several different digestion conditions was performed in order to increase our knowledge of the rat liver proteome, especially important to drug metabolism and toxicology. A parallel (combined) tryptic-peptic digestion yielded best overall performance, when mass spectral data were acquired separately and combined prior to database searching. This large-scale data set will be available publicly on the ProteomeXchange server and will therefore be accessible to a large scientific community interested in using this data for their own studies. One of the main goals of this study is to identify a comprehensive list of proteins for follow-up protein covalent binding studies related to drug toxicity.
采用离子交换分级与反相色谱相结合的系统方法，结合高分辨四极杆飞行时间质谱法，优化大鼠肝微粒体的分析。对几种条件进行了比较，以选择最有效的溶解和蛋白水解方案，以实现最高的蛋白质组覆盖率。通过 SDS 和加热来增加微粒体样品的溶解度，从而获得最佳的胰蛋白酶消化条件，与应用 SDS 和加热之前相比，鉴定的蛋白质从 621 种增加到 686 种。胃蛋白酶消化产生了互补的结果，特别是在疏水环境方面，因此可以大大增加序列覆盖率。测试了几种双消化策略，胰蛋白酶和胃蛋白酶串联或并联组合。胰蛋白酶和胃蛋白酶的平行双消化，结合单酶消化的质谱数据，在鉴定的蛋白质数量方面取得了最佳结果，比最佳单酶方法增加了 29%，所有鉴定的蛋白质的序列覆盖率平均提高了 5%。使用我们完整的数据集，共鉴定到 1095 种蛋白质，假阳性率低于 1%，其中 213 种蛋白质（19.5%）为完整膜蛋白。蛋白质组学数据已被提交到 ProteomeXchange 联盟，数据集标识符为 PXD000128。
通过使用几种不同的消化条件的 2D-LC-MS/MS 对大鼠肝微粒体的蛋白质组学分析进行了系统的优化，以增加我们对大鼠肝蛋白质组的了解，这对于药物代谢和毒理学尤为重要。当分别获得质谱数据并在数据库搜索前组合时，平行（联合）胰蛋白酶-胃蛋白酶消化产生了最佳的整体性能。这个大规模数据集将在 ProteomeXchange 服务器上公开提供，因此对有兴趣将这些数据用于自己研究的广大科学界是可用的。本研究的主要目标之一是确定一个全面的蛋白质列表，用于与药物毒性相关的后续蛋白质共价结合研究。
