Kinetic Size-Exclusion Chromatography with Mass Spectrometry Detection: An Approach for Solution-Based Label-Free Kinetic Analysis of Protein鈥揝mall Molecule Interactions

详细信息    查看全文

• 作者：Jiayin Bao ; Svetlana M. Krylova ; Leonid T. Cherney ; J. C. Yves LeBlanc ; Patrick Pribil ; Philip E. Johnson ; Derek J. Wilson ; Sergey N. Krylov
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：October 21, 2014
• 年：2014
• 卷：86
• 期：20
• 页码：10016-10020
• 全文大小：349K
• ISSN：1520-6882

文摘

Studying the kinetics of reversible protein鈥搒mall molecule binding is a major challenge. The available approaches require that either the small molecule or the protein be modified by labeling or immobilization on a surface. Not only can such modifications be difficult to do but also they can drastically affect the kinetic parameters of the interaction. To solve this problem, we present kinetic size-exclusion chromatography with mass spectrometry detection (KSEC-MS), a solution-based label-free approach. KSEC-MS utilizes the ability of size-exclusion chromatography (SEC) to separate any small molecule from any protein鈥搒mall molecule complex without immobilization and the ability of mass spectrometry (MS) to detect a small molecule without a label. The rate constants of complex formation and dissociation are deconvoluted from the temporal pattern of small molecule elution measured with MS at the exit from the SEC column. This work describes the concept of KSEC-MS and proves it in principle by measuring the rate constants of interaction between carbonic anhydrase and acetazolamide.