Department of Chemistry, Institute of Organic Chemistry, University of Cologne, Greinstrasse 4, D-50939 Cologne, Germany.
J Mass Spectrom. 2010 Feb;45(2):178-89. doi: 10.1002/jms.1702.
Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-linked 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.
化学交联结合随后的酶解和创建的交联产物的质谱分析，提供了一种评估低分辨率蛋白质结构并深入了解蛋白质界面的替代方法。在本研究中，我们报告了一种基于 Edman 降解化学的创新交联剂的设计，该交联剂在电喷雾电离（ESI）-串联质谱（MS/MS）产物离子质谱中导致特征质量转移的片段离子和恒定中性损失（CNL）的形成，从而能够明确鉴定交联肽。此外，特征性的中性损失反应有助于通过适合高通量研究的多重反应监测进行自动分析，具有良好的灵敏度和选择性。新型交联剂的功能依赖于硫脲部分中高亲核性硫的存在，可有效进行分子内攻击，从而预测并优先裂解甘氨酰-脯氨酰酰胺键。我们的创新分析概念和碰撞诱导解离化学交联试剂的多功能适用性，通过神经肽 SP、促黄体激素释放激素 LHRH 和溶菌酶进行了示例说明。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具有广泛的应用。
