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内在的N-终端反应性和DSSO-碳酸盐和碳酸盐基交叉连接器的改进分析

Alessio Di Ianni^1,2,3, Thomas Fabian Leischner⁴, Bogdan-Razvan Brutiu⁴

¹Center for Structural Mass Spectrometry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale D-06120, Germany.

Analytical chemistry

|March 11, 2026

概括

新的NHS碳酸盐交叉连接器为交叉连接质谱 (XL-MS) 提供了增强的稳定性和独特的N端反应性. 这些试剂提高了交叉连接的效率,并提供了互补的结构洞察力,特别是对于N端相互作用.

科学领域:

生物化学生物化学
化学生物学化学生物学
蛋白质组学是指蛋白质组学.

背景情况:

交联质谱 (XL-MS) 对于研究蛋白质结构和动态至关重要.
传统的交叉链接器使用N-hydroxysuccinimide (NHS) Ester,主要针对氨酸残留物.
现有交叉连接器的有限反应性和稳定性可能会阻碍全面的结构分析.

研究的目的:

为了优化NHS基于碳酸盐的交叉链接器的溶液中的反应性和稳定性.
为了描述dsuccinimidyl sulfoxide carbamate (DSSO-carbamate) 的气相解离行为.
在XL-MS中研究NHS碳酸盐与NHS Ester相比,N-终端向偏好.

主要方法:

优化DSSO-碳酸盐,DSSO的NHS碳酸盐类似物.
气相解离的表征和对DSSO-carbamate的碰撞能量的优化.
使用DSSO-carbamate,DSBU和NNP9对牛血清白蛋白和α-synuclein进行的比较交叉链接实验.
全蛋白质组的XL-MS分析.

主要成果:

与NHS相比,DSSO-carbamate表现出增强的稳定性和改进的交叉链接效率.
在NHS的碳酸盐交叉链接器中,对交叉链接蛋白N-termini的倾向显著更高.
对比研究证实了NHS碳酸盐在不同试剂和蛋白质的N端选择性.

结论:

基于碳酸盐的NHS交叉链接器提供了补充XL-MS数据,特别是用于研究N端蛋白相互作用.
这些试剂提供了增强的稳定性和独特的选择性,在生物结合和化学蛋白质组学中扩大了应用.
对于NHS碳酸盐的N-终端偏好为研究功能相关的N-终端修饰和相互作用提供了有价值的工具.

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