Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?
Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?
复制标题
DOI：
10.1021/acs.analchem.9b02075
复制
发表时间：
2019-10-01
影响因子：
7.4
通讯作者：
Cooper, Helen J.
中科院分区：
文献类型：
--
作者：
Illes-Toth, Eva;Cooper, Helen J.
天然环境质谱法具有同时分析薄组织切片内的天然蛋白质结构和空间分布的潜力。尽管灵敏度高，但原则上可以获得存在的任何蛋白质的该信息，而不需要蛋白质身份的先验知识。到目前为止，自然环境质谱法主要利用液体萃取表面分析（LESA）采样技术。在这里，我们解决了一个基本的问题：观察到的蛋白质结构后，天然液体提取表面分析的蛋白质结构的基板内的代表，或提取过程中促进重折叠（或展开）？具体来说，我们的目的是确定是否蛋白质-配体复合物后观察到LESA指示复合物存在于基板，或人工制品的采样过程。研究的系统是肌红蛋白及其非共价结合血红素辅因子，锌结合蛋白碳酸酐酶及其与乙氧唑胺的结合。电荷态分布，漂移时间曲线，和碰撞截面测定液体萃取表面分析离子迁移率质谱的天然和变性蛋白质，并与直接输液电喷雾得到的。结果表明，它是不可能的再折叠变性的蛋白质与伴随的配体结合（既不是血红素，锌，也不是乙氧唑胺）简单地通过使用天然样LESA溶剂。也就是说，蛋白质-配体复合物仅在存在于底物中时通过LESA MS观察到。
Native ambient mass spectrometry has the potential for simultaneous analysis of native protein structure and spatial distribution within thin tissue sections. Notwithstanding sensitivity, this information can, in principle, be obtained for any protein present with no requirement for a priori knowledge of protein identity. To date, native ambient mass spectrometry has primarily made use of the liquid extraction surface analysis (LESA) sampling technique. Here, we address a fundamental question: Are the protein structures observed following native liquid extraction surface analysis representative of the protein structures within the substrate, or does the extraction process facilitate refolding (or unfolding)? Specifically, our aim was to determine whether protein-ligand complexes observed following LESA are indicative of complexes present in the substrate, or an artifact of the sampling process. The systems investigated were myoglobin and its noncovalently bound heme cofactor, and the Zn-binding protein carbonic anhydrase and its binding with ethoxzolamide. Charge state distributions, drift time profiles, and collision cross sections were determined by liquid extraction surface analysis ion mobility mass spectrometry of native and denatured proteins and compared with those obtained by direct infusion electrospray. The results show that it was not possible to refold denatured proteins with concomitant ligand binding (neither heme, zinc, nor ethoxzolamide) simply by use of native-like LESA solvents. That is, protein-ligand complexes were only observed by LESA MS when present in the substrate.