Electron capture dissociation (ECD) is an ion activation strategy that involves ions interacting with low-energy electrons to induce fragmentation. For proteins, the electron capture process causes the N-Cα bond to fragment, producing c/z-type fragments, in comparison to the b/y-type fragments typically obtained by CID. Because these fragments generally come from flexible and solvent-exposed regions, the hydrophobic core is often left intact after the ECD process. This can provide information about the gas phase structures of native-like protein complexes. Historically, this technique was limited to FTICR instruments due to the feasibility of trapping electrons in the ICR cell to interact with trapped protein cations. When combined with long trapping times, FTICR-based ECD experiments on small proteins revealed that, on long time scales, proteins can elongate and collapse in ways that affect ECD fragmentation. While those studies took place in an FTICR instrument, the Ruotolo lab recently illustrated that ECD could also be sensitive to source-induced structural changes on an IM-Q-TOF instrument. The work presented here examines a recently developed cyclic IMS QTOF platform that features commercially available SID and ExD devices. We demonstrate which pre-IM regions can be prone to activating native-like proteins and protein complexes, and make recommendations on how to tune those regions. We also demonstrate how isCA can result in quaternary structure rearrangement and how SID can be a sensitive diagnostic tool of that effect. While isCA -fixed SID experiments were performed by the Wysocki lab in the past, those experiments demonstrated the effect of isCA on SID through large jumps between isCA energies. For the first time, this work demonstrates the same effect, in greater detail, by varying isCA in small steps while tracking the effect on the SID pathways for standard protein complexes, while also reporting protein complex drift time changes and monomer charge states, during the isCA -fixed SID experiments. Overall, we illustrate that SID, in particular, and ECD, can serve as independent means to validate an instrument's source tuning to strike the balance between maximum signal intensity and adduct removal, while maintaining native-like structure for protein complexes. Panel C was adapted with permission from reference 32, copyright 2022 American Chemical Society. Ubiquitin from bovine erythrocytes was purchased from Sigma Aldrich (Ubq, monomer, 8.5 kDa, P/N U6253), streptavidin (SA, tetramer, 53 kDa) was purchased from Thermo Scientific (catalog number 21125), human recombinant C-reactive protein (CRP, pentamer, 115 kDa) was purchased from EMD Millipore (P/N 236608), and phosphorylase B (PhosB, dimer, 197 kDa) was purchased from Millipore Sigma (P/N P6635). Tryptophan RNA-binding attenuation protein (TRAP) from Bacillus stearothermophilus (bst) was obtained from Prof. Mark Foster's lab at The Ohio State University. It was expressed and purified as previously described, with the minor alteration that cell lysing was accomplished via sonication after overnight expression. Proteins purchased as lyophilized powder were reconstituted in 18 MΩ water to a protein complex concentration of ~40 µM, except for Ubq, which was reconstituted to 100 µM. All proteins were buffer exchanged twice into 200 mM ammonium acetate using BioRad Microspin 6 buffer exchange columns and diluted to 1 µM for CRP, 2 -3 µM for SA, and 4 uM for PhosB in 160/40 ammonium acetate/triethylammonium acetate, except for Ubq which was diluted to 10 µM with 200 mM ammonium acetate. TRAP was provided at a protein complex concentration of ~1.5 µM in 600 mM ammonium acetate. The 21+ charge state was obtained by spraying from this stock solution. The 15+ charge state was obtained by adding 1 µL of TEAA to 4 µL of the stock solution. For all complexes, the triethylammonium acetate (Sigma Aldrich, P/N 90358) was added for chemical charge reduction because lower charge states often give native-like fragmentation patterns compared to normal charge states from 200 mM ammonium acetate. Ions were introduced into the instrument via nanoelectrospray ionization, using glass capillaries (Sutter Instruments) pulled on a Sutter Instruments P-97 tip puller (Novato, CA). Electrospray voltages were ramped up to 1.3 kV to induce spray, then were ramped down to 0.7 -1.0 kV to sustain spray.