Roughhousing with Ions: Surface-Induced Dissociation and Electron Capture Dissociation as Diagnostics of Q-Cyclic IMS-TOF Instrument Tuning Gentleness

Native mass spectrometry can characterize a range of biomolecular features pertinent to structural biology, including intact mass, stoichiometry, ligand-bound states, and topology. However, when an instrument’s ionization source is tuned to max-imize signal intensity or adduct removal, it is possible that the biomolecular complex’s tertiary and quaternary structures can be rearranged in a way that no longer reflect its native-like conformation. This could affect downstream ion activation exper-iments, leading to erroneous conclusions about the native-like structure. One such activation strategy is surface-induced dis-sociation (SID), which generally causes native-like protein complexes to dissociate along the weakest subunit interfaces, re-vealing critical information about the complex’s native-like topology and subunit connectivity. If the quaternary structure has been disturbed then the SID fingerprint will shift as well. Thus, SID was used diagnose source-induced quaternary struc-ture rearrangement and help tune an instrument’s source and other upstream transmission regions to strike the balance be-tween signal intensity, adduct removal, and conserving the native-like structure. Complementary to SID, electron-capture dissociation (ECD) can also diagnose rearranged quaternary structures and was used after in-source activation to confirm that the subunit interfaces were rearranged, opening the structure to electron capture and subsequent dissociation. These results provide a valuable guide for new practitioners to native mass spectrometry and highlight the importance of using standard protein complexes when tuning new instrument platforms for optimal native mass spectrometry performance.