Structural Comparison of Denatured and Supercharged Proteins in Gas and Solution Phases

Abstract

Mass spectrometry has become a popular tool for studying large biomolecules due to its high sensitivity. Mass spectrometry techniques like ion mobility separation and fragmentation methods allow for top-down analysis to determine protein structures. As a gas phase technique, it is crucial to understand how ions fold or unfold in the gas phase without the support of solvents/water, as well as how they behave during the various energy and pressure changes experienced throughout the instrument. It is especially important to understand how protein gas phase ions behave and to monitor any major structural changes occurring since protein structure directly influences protein function. There is a plethora of structural studies that aim to retain native protein structure without additives or the disruption of native salt bridges and inter- and intra- molecular bonds. However, more work is needed on non-native proteins. This work analyzes denatured and supercharged proteins using different mass spectrometry techniques to compare the solution and gas phase structures. First, measuring the amount of unfolding under various denaturing and instrument conditions helps to identify how denatured protein ions behave in the gas phase without the presence of solvent. Next, comparing denatured structures using solution and gas phase crosslinking identifies specific structural changes as proteins unfold, as well as tracks how well protein denaturation retains solution phase structure in the gas phase. Finally, protein supercharging helps to enhance mass spectrometry analysis by shifting charge state distributions towards a lower m/z range. By comparing a supercharged protein’s ATDs and CCS values, we can measure any influence supercharging has on protein structure. Crosslinking supercharged proteins also allows for a comparison of structure and can help confirm how supercharging agents add charge to proteins. Similar structures between solution and gas phases help to substantiate mass spectrometry as a robust tool for proteomic studies. Structural differences between the two phases facilitate the interpretation of gas phase phenomena for mass spectrometry analysis and highlights how solvent influences protein structure.