In general operation, the "TIMS In" pressure, which is the TIMS cartridge entrance pressure, was set to 2.2 mbar. The "Accumulation time" and "Ramp Time" of the TIMS cartridge were adjusted to 100.0 ms and 1,000.0 ms (10.00 % Duty Cycle), respectively. The "Funnel 1 RF" was 300.0 volt peak-to-peak (Vpp). The "Collision Cell In", which determines the m/z transferring range, was 150.0 V. The "Pre Pulse Storage", a delay for collecting ions between "Transfer Time" and "TOF Pulser On", was 35.0 µs. The "Transfer Time" is the time corresponding to transmitting ions from the collision cell to the TOF stage and was set to 140.0, 320.0, and 520.0 µs depending on the size of the protein complex. Increasing the "Transfer Time" can transfer larger ions to the TOF. The "Collision Gas Flow Rate" was 35.0 % for the ESI Tuning Mix (Agilent, Santa Clara, CA) and 88.0 % for protein complexes. The "Delta Values (Δt1 to Δt6)", shown on Figure , were used to control the TIMS cartridge. The Δt2, Δt4, and Δt6 were change from -120, 100, and 100 V to -90, 70, and 10 V, respectively, for nMS experiments. More details of the typical settings for nMS can be found in the Supporting Information (Table ).