Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process
Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process
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基于膜蛋白富集和消化工艺优化寻找缺失蛋白
DOI：
10.1021/acs.jproteome.6b00389
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发表时间：
2016
影响因子：
4.4
通讯作者：
Xu Ping
中科院分区：
文献类型：
--
作者：
Zhao Mingzhi;Wei Wei;Cheng Long;Zhang Yao;Wu Feilin;He Fuchu;Xu Ping
以MCF7细胞为研究对象，首次建立了一种超速离心法和洗涤剂提取法相结合的膜蛋白富集法。比较了洗涤剂溶液消解和增强型滤膜辅助制样(EFASP)与耗时较长的凝胶内消解方法。在溶液中消化策略中，eFASP与RapiGest相结合鉴定了1125种膜蛋白。类似地，eFASP结合脱氧胆酸钠鉴定了1069个膜蛋白；然而，凝胶内消化鉴定了1091个膜蛋白。5种酶切方法共鉴定出1390个膜蛋白，其中1345个膜蛋白含有≥-2，这是≥-7细胞系乃至乳腺癌组织中最大的膜蛋白数据集。有趣的是，我们鉴定了13个独特的多肽，分别属于8个缺失蛋白(MPS)。最后，通过人工合成的多肽对8个独特的多肽进行了验证。两个蛋白质被确认为MPS，另外两个蛋白质为候选检测。
A membrane protein enrichment method composed of ultracentrifugation and detergent-based extraction was first developed based on MCF7 cell line. Then, in-solution digestion with detergents and eFASP (enhanced filter-aided sample preparation) with detergents were compared with the time-consuming in-gel digestion method. Among the in-solution digestion strategies, the eFASP combined with RapiGest identified 1125 membrane proteins. Similarly, the eFASP combined with sodium deoxycholate identified 1069 membrane proteins; however, the in-gel digestion characterized 1091 membrane proteins. Totally, with the five digestion methods, 1390 membrane proteins were identified with ≥1 unique peptides, among which 1345 membrane proteins contain unique peptides ≥2. This is the biggest membrane protein data set for MCF7 cell line and even breast cancer tissue samples. Interestingly, we identified 13 unique peptides belonging to 8 missing proteins (MPs). Finally, eight unique peptides were validated by synthesized peptides. Two proteins were confirmed as MPs, and another two proteins were candidate detections.