# Mass spectrometry detects folding intermediates populated during urea-induced protein denaturation

## TL;DR

This study shows how mass spectrometry can detect protein folding intermediates in high urea solutions, revealing details missed by traditional methods.

## Contribution

The use of nano ESI emitters allows ESI-MS to analyze proteins in up to 8 M urea, enabling detection of folding intermediates.

## Key findings

- Nano ESI-MS resolves protein charge states in high urea concentrations.
- Structural changes in response to pH and ligand binding are directly observed.
- Intermediate folding states are detected in both simple and complex proteins.

## Abstract

Protein folding stability can be probed using urea, a chaotropic agent that disrupts non-covalent interactions at molar concentrations. The denaturation process is typically monitored via optical spectroscopy, which provides ensemble-averaged measurements and may struggle to resolve folding intermediates. In contrast, electrospray ionization mass spectrometry (ESI-MS) captures a non-averaged snapshot of all populated assembly and folding states within a protein conformational ensemble. However, high urea concentrations have traditionally been considered incompatible with ESI. Here, we leverage recent advancements in nano ESI emitter design, utilizing well-defined small-diameter emitters which enables protein charge states to be resolved from solutions containing up to 8 M urea. This approach allows us to directly detect the disruption of native tertiary and quaternary structures and to monitor stability changes in response to solution pH and ligand binding. We demonstrate this using single-domain proteins that follow simple two-state unfolding pathways, as well as more complex multidomain proteins and multimeric protein complexes. Our results show strong agreement with conventional urea–denaturation curves obtained via optical spectroscopy, while also providing enhanced resolution of intermediate folding and assembly states that are challenging to capture using traditional methods.

Nano ESI enables direct detection of protein folding and assembly states in solutions containing up to 8 M urea, revealing structural intermediates and stability changes inaccessible to traditional spectroscopy approaches.