All samples were loaded into in-house pulled borosilicate glass capillaries and ionized using a custom Bruker nanoelectrospray ionization source. The ionization is performed by bringing a platinum wire into contact with the sample solution and setting the capillary voltage to (0.8 -1.0 kV). The instrument was mass calibrated using ESI Tuning Mix. The SID experiments were conducted with or without single charge state isolation using the quadrupole, as noted in the Results section, and the SID potentials were varied appropriately to initiate the fragmentation of different sizes of chosen analytes (Tuning and operation of the SID device is shown in Table and). Previously, cesium iodide and Agilent ESI Tuning Mix has been reported to use as calibrant for TIMS to calculate collision cross-sections (CCSs) of protein complexes. Here, the protein complexes' CCSs were calculated using the measured mobilities (K) determined after calibrating the TIMS cartridge with ESI Tuning Mix under experimental conditions. The typical acquisition time for lower m/z complexes was 2 to 5 minutes. For HBV and GroEL SID results, the acquisition time was 30 minutes. Although 0.5 minutes of acquisition time was sufficient to identify the SID products, with longer acquisition times, the spectra have a higher signal-to-noise ratio (S/N), which provides higher quality spectra (Figure ). The instrument was operated using Bruker's otofControl 6.2 software, and the data were analyzed using DataAnalysis 5.3. The center and full width half maximum height (FWHM) of peaks were determined using MATLAB R2020a. The single peak resolution was defined as the ratio of the peak center location divided by its FWHM for both ion mobility ( IM Rp, (1/K0)/Δ(1/K0)) and m/z ( m/z Rp, m/z/Δm/z).