Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

详细信息    查看全文

• 作者：Claire M. Ouimet ; Hao Shao ; Jennifer N. Rauch ; Mohamed Dawod ; Bryce Nordhues ; Chad A. Dickey ; Jason E. Gestwicki ; Robert T. Kennedy
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：August 16, 2016
• 年：2016
• 卷：88
• 期：16
• 页码：8272-8278
• 全文大小：386K
• 年卷期：0
• ISSN：1520-6882

文摘

Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein–protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody–antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme–antibody interaction was found to have K_d = 24 ± 3 nM by PXCE and K_d = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have K_d = 25 ± 5 nM by PXCE which agrees with K_d values reported without cross-linking. Hsp70–Bag3 binding site mutants and small molecule inhibitors of Hsp70–Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC₅₀ values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.