Collision-Induced Unfolding of High-

Native mass spectrometry (nMS) is a powerful tool for the rapid characterization of protein ions and protein-ligand complexes. By coupling nMS with ion mobility spectrometry (IMS), and collisional activation, we can rapidly obtain insights into protein conformation, and stability can be rapidly obtained. Originally incapable of this workflow, recent work enabled this collision-induced unfolding (CIU) process on commercially available Bruker timsTOF instruments. This early work, however, faced challenges in transmitting larger proteins and sought to unfold only small proteins up to 29 kDa. In this study, we continue the development of this technique and optimized instrument settings to enable the transmission of proteins up to 8,000 Th. The technique also demonstrates the capability to sufficiently energize ions to unfold native-like dimers of superoxide dismutase and β-lactoglobulin and the 45 kDa monomeric ovalbumin. When this TIMS activation technique is applied to large protein ions, however, limited unfolding was observed for bovine serum albumin, and no unfolding was observed for immunoglobulin G likely reflecting the limit of activation for this workflow.