Depicted in Figure are calibrated collision cross-section (CCS) distributions for Ubq 6+ (CCS calibration was achieved using Agilent ESI Tuning Mix as the calibrants, with IMSCal software). Panel A features the hottest (most activating) tuning, and panel F is the coldest (least activating) tuning, based on matches with CCS in the literature for Ubq. The relevant instrument parameters are listed in the figure. As the helium cell entrance, bias, and exit were tuned to have lower voltage deltas, the CCS distribution shifted from extended conformations between 1250 -1750 Å 2 to slightly more compact conformations between 1000 -1500 Å 2 . As the post-Trap bias was lowered from 35 V to 20 V the CCS distribution shifted to favor the most compact conformational state, centered around 1100 Å 2 . When the StepWave body and head gradient deltas were lowered to 1 V apiece, a slight shift towards the most compact conformation is seen, though not nearly as dramatic of a shift as was seen in lowering the deltas and bias of the helium cell and post-Trap bias, respectively. It is possible that this slight shift could result from the larger, slower Ubq conformer being scattered in the He cell and not arriving at the detector. However, this could also affect the slower 5+ charge state as well, which is not observed when we compare relative intensities for 6+/5+ in the mass spectra under these different tuning conditions. In terms of absolute signal intensity, as the conditions are tuned from Figure to 2F, the mass spectra indicate an approximate 50% signal intensity loss as the instrument conditions become cooler. Despite that loss the spectra are still easily interpretable with appropriate S/N. With minimized deltas in the StepWave and the helium cell, and minimized post-Trap bias, the least activating tune parameters were found for Ubq.