Protein Cross-linking Capillary and Microchip Electrophoresis for Protein-Protein Interaction Analysis

Capillary Electrophoresis;Protein-Protein Interactions;Microfluidics;Chemistry;Science;Chemistry

Ouimet, ClaireRuotolo, Brandon Thomas ;

University of Michigan

关键词: Capillary Electrophoresis;    Protein-Protein Interactions;    Microfluidics;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/147636/cmoui_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Proteins perform their functions as part of multi-protein complexes. These protein complexes are vital for carrying out and regulating cellular processes. As such, there is a need for tools to measure protein-protein interaction (PPI) affinity, stoichiometry, and inhibition in order to map interaction sites and screen for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and stoichiometries. While many techniques exist for PPI analysis they often require large amounts of protein, have relatively low throughput, or have utility among a narrow range of PPIs.Capillary electrophoresis (CE) has demonstrated utility for determination of PPI affinity and for screening of PPI modulators. CE has a number of advantages in PPI analysis including low sample volume requirements, direct detection of complexes, and the potential for high-throughput. However, method development for analysis of PPIs by CE is often hampered by the need to maintain the native interaction during the separation and prevent protein adsorption to the capillary wall. Here protein cross-linking capillary electrophoresis (PXCE) is reported and described. Covalently cross-linking interacting proteins prior to electrophoresis eliminates the need to maintain the native interaction during the separation, facilitating method development. The PXCE method is demonstrated for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Separation of free protein from protein complex is achieved either by using capillary zone electrophoresis or by capillary gel electrophoresis. PXCE is demonstrated to give quantitative results for PPI affinity and inhibition. Next, we expanded the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. A short cross-linking reaction time of 10 s is found to have sufficient yields for a variety of complexes. Factors influencing non-specific cross-linking are also explored with concentrations of protein >20 µM yielding non-specific complexes. Apparent dissociation constants for seven different PPIs spanning from low nanomolar to low micromolar are presented. Good agreement was found to non-cross-linking methods. Assays of point mutations in the protein interaction site and nucleotide state dependence of association are also presented. Protein complexes less than about 250 kDa are accessible using the presented method. Separation time is also reduced to about 1 min/sample.Finally, a method for increasing the throughput of sample analysis is presented. Here, a microchip gel electrophoresis separation allowed for protein separation in 2.5 s. Further, a novel device for removing oil from segmented droplet flow based on density is demonstrated for coupling nanoliter-scale sample droplets to microchip separation for rapid and automated sample analysis. Throughputs of 10 s per sample are achieved with multiple injections made per sample. Utility of this device for application to PPI analysis and enzymatic reactions is presented. Specifically, Hsp70-Bag3 interaction and SIRT5 enzymatic reaction samples were assayed. The results suggest future utility of the device and PXCE method for screening of enzyme and PPI modulators.