Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
Department of Biology, Stanford University, Stanford, CA 94305;
Proc Natl Acad Sci U S A. 2014 Jul 1;111(26):9455-60. doi: 10.1073/pnas.1320298111. Epub 2014 Jun 17.
The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.
近年来，使用化学交联结合 MS 鉴定交联肽的方法来研究蛋白质和蛋白质复合物已经得到了越来越广泛的应用。到目前为止，此类分析几乎完全使用同双功能、伯胺反应交联试剂。在这里，我们报告了一种针对羧基的正交交联化学的开发和应用。使用同双功能二酰肼作为交联试剂，在中性 pH 下进行偶联化学，这与大多数蛋白质复合物的结构完整性兼容，从而实现酸性残基的化学交联。除了通过不同间隔长度的二酰肼插入形成交联外，还可以获得零长度交联产物，从而提供额外的结构信息。我们展示了该反应的应用以及用于伴侣蛋白 TRiC/CCT 和 26S 蛋白酶体的交联肽的 MS 鉴定。结果表明，针对酸性残基的交联靶标提供了距离约束，这些约束与赖氨酸交联获得的约束互补且正交，从而扩展了可以从交联研究中获得并用于混合建模方法的结构信息的产量。
