Experiment Type: Optimize Pre-IM Tuning With each new instrument generation, end-users must optimize instrument tuning to meet their experimental needs. For the nMS community, this often means tuning instrument voltages to minimize ion activation in the source and other pre-selection quadrupole areas to maintain native-like biomolecule conformations; and in the post-activation areas to minimize the risk of secondary fragmentation. While the Waters cIM features a unique IM region that can be complex to tune, the Zenobi lab produced a helpful body of work related to that topic. In terms of activation regions, the Waters cIM instrument features multiple places for deliberate activation (Figure ): the cone voltage between the StepWave and the source ion guide, the Trap cell, the IM pre-array, and the Transfer cell. To minimize activation prior to the selection quadrupole, the source region (ion source, StepWave, ion guide) should be carefully tuned to minimize unintentional activation and maintain nativelike protein conformations. Between the selection quadrupole and the cIM, the Trap gradient, post-Trap bias, and helium cell should be tuned to minimize pre-IM activation, or to minimize secondary fragmentation after SID or Trap CID. To tune our instrument, we used Ubq's measured CCS as a probe of pre-IM "hotness," and SID of CRP to fine-tune for native protein complexes. Figure . A -F) Collision cross-section distributions for Ubq 6+. He cell is the helium cell, pTb is the post-Trap bias, and BG and HG are the body gradient and head gradient, respectively, in the source StepWave. The underlined parameters highlight what is varied from one panel to the next. The flame icon indicates the "hottest" tuning profile, resulting in Ubq 6+ adopting extended conformations, while the snowflake indicates the "coldest" tuning profile, resulting in compact conformations. The red to orange to blue peak colors also indicate the transition from hot to cold tuning.