# Investigating the Effect of Isoelectric Points on the Gas-Phase Stability of Native-like Proteins Analyzed in Positive- versus Negative-Ion Mode by IMS-MS

## TL;DR

This study shows that the gas-phase stability of proteins analyzed by IMS-MS depends on the ionization polarity, with proteins being more stable in the polarity that matches their solution-phase charge.

## Contribution

The work reveals that ionization polarity significantly affects the gas-phase stability of native-like proteins in IMS-MS.

## Key findings

- Proteins analyzed in the polarity matching their solution-phase charge are more stable in the gas phase.
- Positive and negative ions show significant differences in gas-phase stability despite similar initial folded structures.
- The energy required for unfolding is higher in the polarity corresponding to the protein's solution-phase charge.

## Abstract

Native ion mobility
spectrometry-mass spectrometry (IMS-MS)
is
routinely used for analysis of folded proteins and protein complexes.
For many proteins, the three-dimensional structure is maintained during
electrospray ionization (ESI) as the protein transitions to the gas
phase, allowing for detailed investigation of the gaseous, ionic protein’s
structure and stability. Much of the native IMS-MS research has been
conducted in positive-ion mode (+ESI), even when the protein of interest
has a net-negative charge in solution at physiological pH. We hypothesize
that analyzing a protein in the polarity that is opposite to its solution-phase
charge, such as analyzing net-negative proteins by +ESI-MS, disrupts
the network of noncovalent-bonding interactions within the protein
to a greater extent than using the polarity that matches the protein’s
solution-phase charge, resulting in differences in protein stability.
Herein, we show that while most protein ions have similar initial,
folded structures in +ESI and negative-ion mode (−ESI), positive
and negative ions exhibit significant differences in gas-phase stability.
Furthermore, the energy required to cause this unfolding is often
greater in the polarity corresponding to the solution-phase charge
of the protein, indicating that the protein is more stable in that
polarity. Thus, this work highlights the necessity of considering
polarity when conducting native IMS-MS experiments.