Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies.
Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies.
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DOI：
10.1021/acs.jproteome.8b00235
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发表时间：
2018-07-06
影响因子：
4.4
通讯作者：
Hummon, Amanda B.
中科院分区：
文献类型：
--
作者：
Ludwig, Katelyn R.;Schroll, Monica M.;Hummon, Amanda B.
关键词：
自下而上的蛋白质组学策略依赖于将蛋白质有效地消化成多肽进行质谱分析。蛋白质组学分析通常采用基于溶液和基于过滤器的策略。近年来，滤膜辅助样品制备(FASP)因其在质谱分析前去除十二烷基硫酸钠的能力而成为主要的基于滤膜的方法。然而，FASP协议的耗时特性导致了新的基于过滤器的策略的发展。悬浮捕捉器(S捕捉器)最近被报道为游离三聚氰胺和溶液中策略的替代方案，因为它们允许在典型的游离三聚氰胺方案的一小部分时间内获得高浓度的十二烷基硫酸钠。在这项研究中，我们比较了从十二烷基硫酸钠和尿素裂解缓冲液中提取的蛋白质在溶液中、游离天冬氨酸和S陷阱消化中的产率。我们进行了无标记定量，以分析使用每种方法鉴定的蛋白质组部分的差异。总体而言，我们的结果表明，每种消化方法在方法类型内都具有高度的重复性。然而，S陷阱提供了最有效的消化和最多数量的独特蛋白质识别，因此优于脂肪酶和溶液中消化。这是第一次提供两种基于过滤器的消化方法和传统的溶液中方法的直接定量比较，以提供关于最有效的蛋白质组制备的信息。
Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in-solution approach to provide information regarding the most efficient proteomic preparation.
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