Institute for Systems Biology, Seattle, Washington 98109, USA.
Mol Cell Proteomics. 2012 Feb;11(2):M111.008318. doi: 10.1074/mcp.M111.008318. Epub 2011 Nov 7.
Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical cross-linking combined with mass spectrometry is an attractive approach for studying protein-protein interactions and protein structure, but to date its use has been limited largely by low yields of informative cross-links (because of inefficient cross-linking reactions) and by the difficulty of confidently identifying the sequences of cross-linked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile cross-linking reagent, BDRG (biotin-aspartate-Rink-glycine), which addresses these issues. BDRG incorporates a biotin handle (for enrichment of cross-linked peptides prior to MS analysis), two pentafluorophenyl ester groups that react with peptide amines, and a labile Rink-based bond between the pentafluorophenyl groups that allows cross-linked peptides to be separated during MS and confidently identified by database searching of their fragmentation spectra. We developed a protocol for the identification of BDRG cross-linked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of cross-linker-modified peptides. Importantly, our approach permits the use of high accuracy precursor mass measurements to verify the database search results. We demonstrate the utility of the approach by applying it to purified yeast TFIIE, a heterodimeric transcription factor complex, and to a single-step affinity-purified preparation of the 12-subunit RNA polymerase II complex. The results show that the method is effective at identifying cross-linked peptides derived from purified and partially purified protein complexes and provides complementary information to that from other structural approaches. As such, it is an attractive approach to study the topology of protein complexes.
蛋白质结构和蛋白质-蛋白质相互作用的知识对于理解生物过程至关重要。化学交联结合质谱分析是研究蛋白质-蛋白质相互作用和蛋白质结构的一种有吸引力的方法，但迄今为止，由于交联反应效率低（导致信息量低的交联产物）以及难以从其碎片谱中自信地鉴定交联肽对的序列，其应用受到了很大限制。在这里，我们提出了一种基于新型 MS 不稳定交联试剂 BDRG（生物素天冬氨酸-赖氨酰-甘氨酸）的方法，该方法解决了这些问题。BDRG 包含一个生物素接头（用于在 MS 分析之前富集交联肽）、两个与肽胺反应的五氟苯基酯基团，以及五氟苯基基团之间不稳定的 Rink 键，允许在 MS 期间分离交联肽，并通过其碎片谱的数据库搜索自信地鉴定。我们开发了一种用于鉴定来自纯化或部分纯化蛋白质复合物的 BDRG 交联肽的方案，包括软件来辅助鉴定不同类别的交联剂修饰肽。重要的是，我们的方法允许使用高精度的前体质量测量来验证数据库搜索结果。我们通过将其应用于纯化的酵母 TFIIE（一种异源二聚体转录因子复合物）和一步亲和纯化的 RNA 聚合酶 II 十二亚基复合物制剂，证明了该方法的实用性。结果表明，该方法有效地鉴定了来自纯化和部分纯化蛋白质复合物的交联肽，并提供了与其他结构方法互补的信息。因此，它是研究蛋白质复合物拓扑结构的一种有吸引力的方法。
