Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies.,Journal of Proteome Research

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Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2018-05-24 , DOI: 10.1021/acs.jproteome.8b00235
Katelyn R Ludwig _{1,

2} , Monica M Schroll _{1,

2} , Amanda B Hummon ₂

Affiliation

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.

中文翻译：

用于自下而上蛋白质组学研究的溶液内、FASP 和基于 S-Trap 的消化方法的比较。

自下而上的蛋白质组学策略依赖于将蛋白质有效消化成肽进行质谱分析。溶液内和基于过滤器的策略通常用于蛋白质组学分析。近年来，过滤辅助样品制备 (FASP) 已成为主要的基于过滤器的方法，因为它能够在质谱分析之前去除 SDS。然而，FASP 协议的耗时特性导致了基于过滤器的新策略的开发。悬浮陷阱 (S-Trap) 最近被报道为 FASP 和溶液内策略的替代方案，因为它们允许在典型 FASP 协议的一小部分时间内实现高浓度的 SDS。在本研究中，我们比较了在 SDS 和基于尿素的裂解缓冲液中提取的蛋白质的溶液内、FASP 和 S-Trap 消化的产量。我们进行了无标记量化，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，每种消解方法在该方法类型内都具有高度的重现性。然而，S-Traps 通过提供最有效的消化和最多数量的独特蛋白质鉴定，优于 FASP 和溶液内消化。这是首次对两种基于过滤器的消化方法和传统的溶液内方法进行直接定量比较，以提供有关最有效蛋白质组学制备的信息。通过提供最有效的消化和最多数量的独特蛋白质鉴定，S-Trap 的性能优于 FASP 和溶液内消化。这是首次对两种基于过滤器的消化方法和传统的溶液内方法进行直接定量比较，以提供有关最有效蛋白质组学制备的信息。通过提供最有效的消化和最多数量的独特蛋白质鉴定，S-Trap 的性能优于 FASP 和溶液内消化。这是首次对两种基于过滤器的消化方法和传统的溶液内方法进行直接定量比较，以提供有关最有效蛋白质组学制备的信息。

更新日期：2018-05-25

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