Evaluation and optimization of removal of an acid-insoluble surfactant for shotgun analysis of membrane proteome
Evaluation and optimization of removal of an acid-insoluble surfactant for shotgun analysis of membrane proteome
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DOI：
10.1002/elps.201000161
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发表时间：
2010-08-01
期刊：
影响因子：
2.9
通讯作者：
Liang, Songping
中科院分区：
文献类型：
--
作者：
Lin, Yong;Liu, Yi;Liang, Songping
由于脱氧胆酸钠（SDC）在高浓度下与蛋白酶活性相容，可有效提高膜蛋白的增溶和变性，近年来在膜蛋白质组学研究领域受到越来越多的关注。SDC可以通过酸化，然后离心（即酸沉淀，AP）或用乙酸乙酯萃取（即相转移，PT），以便不干扰下游分析，如LC-MS/MS。在这项研究中，这两种策略进行了系统的评估，比较和优化。研究结果表明，AP和PT两种策略均导致一定量的胰蛋白酶肽损失，而PT策略在去除SDC过程中损失的胰蛋白酶肽更多。然而，损失的肽可以通过分别洗涤AP和PT期间产生的沉淀和固体内容物来大部分回收。通过回收丢失的多肽，显著提高了蛋白质，特别是跨膜蛋白和低丰度蛋白质的鉴定效率。相比之下，通过回收丢失的肽段进行优化后，AP策略不仅能达到与PT策略相当的鉴定效率，而且比PT策略更经济、更安全、更易于操作，因此AP策略上级优于PT策略。
Due to its compatibility with protease activity at high concentration, sodium deoxycholate (SDC) can be used to effectively improve the solubilization and enzymolysis of membrane proteins and has received increasing attention in the field of membrane proteome analysis in recent years. SDC can be removed from digests by means of acidification followed by centrifugation (i.e. acid precipitation, AP) or extraction with ethyl acetate (i.e. phase transfer, PT) so as not to interfere with the downstream analyses like LC-MS/MS. In this study, the two strategies were systematically evaluated, compared and optimized. The results of the study demonstrated that both of the AP and PT strategies led to a certain amount of tryptic peptides being lost, and in PT strategy even more peptides were lost during SDC removal process. However, the lost peptides could be mostly recovered by washing the pellet and solid content produced during AP and PT, respectively. By recovering the lost peptides, the identification efficiency of proteins, especially transmembrane and low abundance ones, was significantly improved. Comparatively, after optimization by recovering the lost peptides, AP strategy was superior to PT strategy because the former not only could achieve the comparable identification efficiency with the latter but also was more economical, safer and easier to operate than the latter.