Combined chemical modification and collision induced unfolding using native ion mobility‐mass spectrometry provides insights into protein gas‐phase structure

Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas-phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility-mass spectrometry (IM-MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas-phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N-terminus and E22 in a ubiquitin binding protein UBA2.