All experiments were performed on Waters Synapt G2 HDMS mass spectrometer in denaturing conditions using NanoLockSpray ion source for offline nanoESI. Each ubiquitin dimer was diluted to a final concentration of 10 uM in 49/50/1% (v/v %) water/methanol/formic acid. Applied instrument conditions: capillary voltage -1.0 kV, source temperature -20°C, sampling cone -30 V, extraction cone -1.8 V, trap gas flow -3 mL/min, helium cell gas flow -180 mL/min, IMS gas flow -80 mL/min. TWIMS parameters were adjusted to receive the most efficient separation between ubiquitin dimers at highest available wave height -40 V. CCS calibration was performed by adding 5 uM apo-myoglobin (Sigma-Aldrich, CAS Number 9008-45-1) as an internal standard.