# Facile detection of peptide–protein interactions using an electrophoretic crosslinking shift assay

## TL;DR

This paper introduces a new method called ECSA to detect weak protein-peptide interactions, which are important for understanding cellular functions.

## Contribution

The novel contribution is the development of the electrophoretic crosslinking shift assay (ECSA) for detecting low-affinity domain-motif interactions.

## Key findings

- ECSA successfully detects micromolar interactions using minimal protein input.
- The method is effective for studying short linear motifs in protein interactions.
- ECSA bridges high-throughput screening and detailed structural analysis.

## Abstract

Protein–protein interactions with high specificity and low affinity are functionally important but are not comprehensively understood because they are difficult to identify. Particularly intriguing are the dynamic and specific interactions between folded protein domains and short unstructured peptides known as short linear motifs. Such domain–motif interactions (DMIs) are often difficult to identify and study because affinities are modest to weak. Here we describe “electrophoretic crosslinking shift assay” (ECSA), a simple in vitro approach that detects transient, low affinity interactions by covalently crosslinking a prey protein and a fluorescently labeled bait. We demonstrate this technique on the well characterized DMI between MAP kinases and unstructured D-motif peptide ligands. We show that ECSA detects sequence-specific micromolar interactions using less than a microgram of input prey protein per reaction, making it ideal for verifying candidate low-affinity DMIs of components that purify with low yield. We propose ECSA as an intermediate step in SLiM characterization that bridges the gap between high throughput techniques such as phage display and more resource-intensive biophysical and structural analysis.