Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes
Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes
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DOI：
10.1073/pnas.1320298111
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发表时间：
2014-07-01
影响因子：
11.1
通讯作者：
Aebersold, Ruedi
中科院分区：
文献类型：
--
作者：
Leitner, Alexander;Joachimiak, Lukasz A.;Aebersold, Ruedi
近年来，使用化学交联随后通过MS鉴定交联肽的蛋白质和蛋白质复合物的研究已经发现越来越广泛的用途。到目前为止，这种分析几乎完全使用同双功能的胺反应性交联试剂。在这里，我们报告了针对羧基的正交交联化学的开发和应用。酸性残基的化学交联是使用同双功能二酰肼作为交联试剂和在中性pH下的偶联化学实现的，该偶联化学与大多数蛋白质复合物的结构完整性相容。除了通过插入具有不同间隔子长度的二酰肼形成的交联之外，还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对伴侣蛋白TRiC/CCT和26 S蛋白酶体产生的交联肽的MS鉴定。结果表明，交联的酸性残基的靶向提供的距离限制，是互补的和正交的那些从赖氨酸交联，从而扩大产量的结构信息，可以从交联研究和混合建模方法中使用。
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.