Collision-Induced Unfolding of High-m/z Native-like Protein Ions within a Trapped Ion Mobility Spectrometer

Native mass spectrometry (nMS) is a powerful tool for the characterization of proteins and protein-ligand complexes. By coupling nMS with ion mobility spectrometry (IMS), and collisional activation, insights into protein conformation and stability can be rapidly obtained. Originally incapable of this workflow, recent work enabled this collision-induced unfolding (CIU) workflow on commercially available Bruker timsTOF instruments. This early work, however, faced challenges in transmit-ting larger proteins and only sought to unfold small proteins up to 29 kDa. In this study, we continue the development of this technique and optimized instrument settings, enabling the ionization and transmission of proteins up to 8,000 Th, and successfully unfolded proteins such as superoxide dismutase, β -lactoglobulin, and ovalbumin on a timsTOF. When this TIMS activation technique is applied large protein ions however, limited unfolding was observed for bovine serum albumin and no unfolding was observed for immunoglobulin G.