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.

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

“…2d ). Thus, we confirm SIFTER maintains endogenous protein complexes without Joule heating with ~100× faster fractionation than in a slab gel, 100−1000× higher sample throughput than a capillary (or comparable to automated capillary systems 51 ), and without purifying, labeling, or crosslinking of complexes 52 .…”

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

“…2d). Thus, we confirm SIFTER maintains endogenous protein complexes without Joule heating with ~100× faster fractionation than in a slab gel, 100-1000× higher sample throughput than a capillary (or comparable to automated capillary systems 47 ), and without purifying, labeling or crosslinking of complexes 48 .…”

Label-free methods for opticalin vitrocharacterization of protein–protein interactions

“…74 The same group further developed this method with an accelerated cross-linking method, which increased throughput to 1 min per sample and showed its validity using 5 different protein-protein interactions. 65 For the Hsp70-HOP interaction, they obtained an electropherogram (left) and saturation binding curve (right) by 10 s glutaraldehyde cross-linking (Fig. 3c).…”