Reactivity and Applications of New Amine Reactive Cross-Linkers for Mass Spectrometric Detection of Protein-Protein Complexes
Reactivity and Applications of New Amine Reactive Cross-Linkers for Mass Spectrometric Detection of Protein-Protein Complexes
复制标题
DOI：
10.1021/ac901651r
复制
发表时间：
2010-01-01
影响因子：
7.4
通讯作者：
Zenobi, Renato
中科院分区：
文献类型：
--
作者：
Bich, Claudia;Maedler, Stefanie;Zenobi, Renato
蛋白质的化学交联允许通过引入共价键稳定非共价复合物。一个至关重要的挑战是找到最快，最有效的交联剂，以最大程度地减少反应时间并处理精致的复合物。通过将N-羟基苯胺，羟苯二唑三唑和1-羟基-7-亚恰亚曲亚唑作为离开基团而不是常用的N-羟基糖核糖核苷的离开，可以合成新的交联链。通过使用基质辅助激光解吸电离（MALDI）质谱法，然后将这些新的交叉链接器与商业上可用的二核酰亚胺suberate（DSS）进行了比较，以共价稳定。抗体 - 抗原复合物。它们显示出更好的效率，产生的交联复合物高约30％，反应速度比DSS快10倍。与GST二聚体的反应用于获取有关其反应效率和动力学的信息。通过将蛋白质GST和泛素的混合物孵育来验证它们仅稳定特定蛋白质复合物的能力。最后，将交联剂与合成肽一起孵育，以研究与各种氨基酸侧链结合的选择性。不仅赖氨酸，而且还发现酪氨酸与含有1-羟基-7-亚恰亚曲亚唑的新合成的交联反应反应。
Chemical cross-linking of proteins permits the stabilization of noncovalent complexes through introduction of covalent bonds. A crucial challenge is to find the fastest and most efficient cross-linkers in order to minimize reaction times and to handle delicate complexes. New cross-linkers were synthesized by introducing N-hydroxyphthalimide, hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole as leaving groups instead of the commonly used N-hydroxysuccimidyl moiety. With the use of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, these new cross-linkers were then compared with the commercially available disuccinimidyl suberate (DSS) for covalent stabilization of the gluthatione-S-transferase (GST) dimer and of an antibody-antigen complex. They showed a better efficiency, generated about 30% more cross-linked complex, and reacted about 10 times faster than DSS. The reaction with the GST dimer was utilized to get information about their reaction efficiency and kinetics. Their ability to stabilize only specific protein complexes was verified by incubating them with a mixture of the proteins GST and ubiquitin. Finally, the cross-linkers were incubated with synthetic peptides to study the selectivity of the binding with various amino acid side chains. Not only lysine but also tyrosine was found to react with the newly synthesized cross-linker containing 1-hydroxy-7-azabenzotriazole as the reactive group.