Collision-induced dissociative chemical cross-linking reagent for protein structure characterization: applied Edman chemistry in the gas phase
Collision-induced dissociative chemical cross-linking reagent for protein structure characterization: applied Edman chemistry in the gas phase
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DOI：
10.1002/jms.1702
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发表时间：
2010-02-01
影响因子：
2.3
通讯作者：
Schaefer, Mathias
中科院分区：
文献类型：
--
作者：
Dreiocker, Frank;Mueller, Mathias Q.;Schaefer, Mathias
化学交联与随后产生的交联产物的酶消化和质谱分析相结合，为评估低分辨率蛋白质结构和洞察蛋白质界面提供了一种替代方法。在这篇文章中，我们报道了一种基于Edman降解化学的创新交联剂的设计，该交联剂能够在电喷雾电离(ESI)-串联质谱仪(MS/MS)的产物离子谱中形成指示质量移位的碎片离子和恒定的中性损失(CNL)，从而能够明确地识别交联肽。此外，特有的中性损失反应便于通过多反应监测实现自动化分析，适合高通量研究，具有良好的灵敏度和选择性。这种新型交联剂的功能依赖于硫脲部分中高度亲核的硫的存在，从而防止有效的分子内攻击导致甘氨酰丙基酰胺键的预测和首选断裂。我们的创新分析概念和碰撞诱导解离化学交联剂的多功能性以P物质、促黄体激素释放激素LHRH和溶菌酶为例。这种新型交联剂有望在探测蛋白质三级结构和研究蛋白质-蛋白质相互作用方面有广泛的应用。版权所有(C)2009 John Wiley&Sons，Ltd.
Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created crosslinked products presents an alternative approach to assess low-resolution protein structures and to gain insight into protein interfaces. In this contribution, we report the design of an innovative cross-linker based on Edman degradation chemistry, which leads to the formation of indicative mass shifted fragment ions and constant neutral losses (CNLs) in electrospray ionization (ESI)-tandem-mass spectrometry (MS/MS) product ion mass spectra, allowing an unambiguous identification of cross-linked peptides. Moreover, the characteristic neutral loss reactions facilitate automated analysis by multiple reaction monitoring suited for high throughput studies with good sensitivity and selectivity. The functioning of the novel cross-linker relies on the presence of a highly nucleophilic sulfur in a thiourea moiety, safeguarding for effective intramolecular attack leading to predictive and preferred cleavage of a glycyl-prolyl amide bond. Our innovative analytical concept and the versatile applicability of the collision-induced dissociative chemical cross-linking reagent are exemplified for substance P, luteinizing hormone releasing hormone LHRH and lysozyme. The novel cross-linker is expected to have a broad range of applications for probing protein tertiary structures and for investigating protein-protein interactions. Copyright (C) 2009 John Wiley & Sons, Ltd.