Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments.
Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments.
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DOI：
10.1021/pr4010019
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发表时间：
2014-04-04
影响因子：
4.4
通讯作者：
Loo, Joseph A.
中科院分区：
文献类型：
--
作者：
Erde, Jonathan;Loo, Rachel R. Ogorzalek;Loo, Joseph A.
定量蛋白质组学实验的完整性取决于蛋白质提取、溶解和消化方法的可靠性和稳健性。去污剂、离液剂和机械破碎的组合可以产生成功的蛋白质制备物;然而，除了样品中已经存在的盐、核酸和脂质之外，随后需要消除这些添加的污染物的方法可能导致显著的样品损失和不完全的污染物去除。最近引入的蛋白质组样品制备方法，过滤器辅助样品制备（FASP），巧妙地规避了许多与传统蛋白质纯化方法相关的挑战，但与显着的样品损失。本文介绍的是一种增强型FASP（eFASP）方法，该方法结合了传统FASP的替代试剂，提高了处理样品的灵敏度、回收率和蛋白质组覆盖率。在eFASP消化过程中用0.2%脱氧胆酸代替尿素提高了细胞溶质和膜蛋白的胰蛋白酶消化效率，还避免了与使用脱氧胆酸钠盐相关的所需的清除步骤。对于经典的FASP，用5%的tetrant-20预钝化Microcon过滤器表面可将肽损失减少300%。一种快速eFASP方法使用三（2-羧乙基）膦和4-乙烯基吡啶在Microcon过滤器上沉积之前烷基化蛋白质，增加烷基化特异性并加速处理。
The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss. Presented here is an enhanced FASP (eFASP) approach that incorporates alternative reagents to those of traditional FASP, improving sensitivity, recovery, and proteomic coverage for processed samples. The substitution of 0.2% deoxycholic acid for urea during eFASP digestion increases tryptic digestion efficiency for both cytosolic and membrane proteins yet obviates needed cleanup steps associated with use of the deoxycholate sodium salt. For classic FASP, prepassivating Microcon filter surfaces with 5% TWEEN-20 reduces peptide loss by 300%. An express eFASP method uses tris(2-carboxyethyl)phosphine and 4-vinylpyridine to alkylate proteins prior to deposition on the Microcon filter, increasing alkylation specificity and speeding processing.
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