Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility‐Mass Spectrometry Provides Insights into Protein Gas‐Phase Structure

Abstract

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.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

“… The fact that proteins lacking basic residues favor the CRM over CEM reveals the limitations of using charge state signatures to assess the distribution of folded states . As a result, proteins with few ionizable groups may display diverging charging patterns upon unfolding. Previous studies have shown that point mutations can affect the CIU pattern of native-like protein ions by modulating their structure in solution. ,,, Our findings reveal that even identically folded proteins can exhibit different CIU fingerprints due to altered surface electrostatics during ESI. Because these differences arise from the locations of ESI charges, not from solution folding, care must be taken when using CIU data to probe solution structures. Gas-phase unfolding is routinely used to identify compounds that can stabilize protein complexes by occupying specific binding pockets. ,− We find that moving charged sites can have a dramatic effect on a protein’s conformation in the gas phase.…”

“… Previous studies have shown that point mutations can affect the CIU pattern of native-like protein ions by modulating their structure in solution. 9 , 15 , 54 , 55 Our findings reveal that even identically folded proteins can exhibit different CIU fingerprints due to altered surface electrostatics during ESI. Because these differences arise from the locations of ESI charges, not from solution folding, care must be taken when using CIU data to probe solution structures.…”

“…Characterizing the interactions, stabilities, and conformations of proteins is of central importance in biochemical and pharmaceutical sciences. Native mass spectrometry (MS) informs about the molecular weight of protein complexes to reveal oligomeric states and ligand binding. − Combined with ion mobility (IM), it can be used to determine collision cross sections (CCS) and identify conformational changes. − If protein complexes are additionally subjected to collisional activation inside the mass spectrometer, the resulting collision-induced unfolding (CIU) can be followed by IM-MS, informing about their gas-phase stabilities. − …”

Optimum collision energies for proteomics: The impact of ion mobility separation

“…53 Separation of oligosaccharides resulted in a resolving power of approximately 120 using three passes, 54 while an estimated resolution of 920 could be achieved after 58 cycles. 55 Because of its outstanding efficiency, cyclic IMS was applied in various proteomics studies as well, both for peptide [56][57][58][59][60][61][62] and protein 27,[63][64][65][66][67][68][69] analysis. This technique was shown to provide more identified peptides resulting in increased sequence coverage.…”

“…64 It was demonstrated that cyclic IMS efficiently separates protein conformers 65,66 and is capable to monitor the extent of protein unfolding. 67,68 Further, protein conformational changes may occur upon (multipass) cyclic experiments depending on the voltage settings applied. 27,69 Establishing that cyclic IMS is indeed a highly relevant platform for our investigations, our aim with the present project is to formulate practical suggestions on choosing the best CE setting for proteomics if IMS is used.…”