Issue 8, 2018

Protein cross-linking capillary electrophoresis at increased throughput for a range of protein–protein interactions

Abstract

Tools for measuring affinities and stoichiometries of protein–protein complexes are valuable for elucidating the role of protein–protein interactions (PPIs) in governing cell functions and screening for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and include stoichiometries from dimers to high order oligomers. Also, most techniques require large amounts of protein which can hamper research for difficult to obtain proteins. Protein cross-linking capillary electrophoresis (PXCE) has the potential to directly measure PPIs and even resolve multiple PPIs while consuming attomole quantities. Previously PXCE has only been used for high affinity, 1 : 1 complexes; here we expand the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. Use of glutaraldehyde as the cross-linking agent was key to advancing the method because of its rapid reaction kinetics. A 10 s reaction time was found to be sufficient for cross-linking and quantification of seven different PPIs with Kd values ranging from low μM to low nM including heat shock protein 70 (Hsp70) interacting with heat shock organizing protein (3.8 ± 0.7 μM) and bcl2 associated anthanogene (26 ± 6 nM). Non-specific cross-linking of protein aggregates was found to be minimal at protein concentrations <20 μM as assessed by size exclusion chromatography. PXCE was sensitive enough to measure changes in PPI affinity induced by the protein nucleotide state or point mutations in the protein-binding site. Further, several interactions could be resolved in a single run, including Hsp70 monomer, homodimer and Hsp70 complexed the with c-terminus of Hsp70 interacting protein (CHIP). Finally, the throughput of PXCE was increased to 1 min per sample suggesting potential for utility in screening.

Graphical abstract: Protein cross-linking capillary electrophoresis at increased throughput for a range of protein–protein interactions

Supplementary files

Article information

Article type
Paper
Submitted
28 Жел. 2017
Accepted
15 Нау. 2018
First published
15 Нау. 2018

Analyst, 2018,143, 1805-1812

Protein cross-linking capillary electrophoresis at increased throughput for a range of protein–protein interactions

C. M. Ouimet, M. Dawod, J. Grinias, V. A. Assimon, J. Lodge, A. K. Mapp, J. E. Gestwicki and R. T. Kennedy, Analyst, 2018, 143, 1805 DOI: 10.1039/C7AN02098H

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements