On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies
On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies
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DOI：
10.1002/rcm.832
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发表时间：
2002-01-01
影响因子：
2
通讯作者：
Roeraade, J
中科院分区：
文献类型：
--
作者：
Benkestock, K;Edlund, PO;Roeraade, J
许多蛋白质和大分子容易形成金属加合物离子，这损害了它们的质谱分析。本研究描述了如何形成不需要的加合物可以最大限度地减少通过在线微透析的非共价结合的研究与低分子量的配体与电喷雾电离质谱（ESI-MS）的大分子。该技术是必不可少的蛋白质配体的研究，由于不需要的加合离子的减少，从而提供了良好的分辨率和灵敏度提高至少5倍。分析系统的核心是一个改进的微透析装置，它是在逆流模式下操作。还提出了一种基于微透析的竞争结合研究的新技术。蛋白质和配体之间的非共价复合物在分析之前在样品瓶中形成。将复合物注射到在线微透析系统中，其中在纤维外部的透析缓冲液中施用竞争性配体。第二配体通过经由透析纤维的壁的运输竞争性地置换第一配体，并且通过ESI-MS检测完整的复合物。
Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The noncovalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS. Copyright (C) 2002 John Wiley Sons, Ltd.