Dobbs Joseph M, Jenkins Meredith L, Burke John E
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada.
Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
J Am Soc Mass Spectrom. 2020 Oct 7;31(10):2202-2209. doi: 10.1021/jasms.0c00283. Epub 2020 Sep 25.
Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for . For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.
从亲和纯化质谱（AP-MS）数据中过滤非特异性结合的污染蛋白是一种成熟的策略，可提高已鉴定蛋白的统计可信度。CRAPome（亲和纯化污染物库）描述了许多纯化策略中存在的污染背景成分。然而，缺乏镍-亚氨基三乙酸（NiNTA）和谷胱甘肽S-转移酶（GST）亲和基质的完整污染物列表。同样，没有（Sf9）污染物可用，并且仅描述了用于的FLAG纯化污染物。对于使用重组蛋白的质谱实验，例如结构质谱实验（氢-氘交换质谱（HDX-MS）、化学交联和自由基足迹），在初始串联质谱（MS/MS）鉴定阶段未能在搜索数据库中包含这些污染物可能会导致肽段鉴定出现问题。我们已经为Sf9和NiNTA或GST纯化策略创建了污染物FASTA数据库，并表明使用这些数据库可以有效提高HDX-MS蛋白覆盖率、片段计数以及肽段鉴定的可信度。这种方法为设计任何纯化方法的污染物数据库提供了一种稳健的策略，这将扩展能够通过HDX-MS进行研究的系统的复杂性。
