Fifty micrograms of protein for each sample was reduced, alkylated and precipitated using methanol/chloroform as previously described and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M urea, 50 mM HEPES pH 7. The TIMS elution voltages were calibrated linearly with three points (Agilent ESI-L Tuning Mix Ions; 622, 922, 1,222 m/z) to determine the reduced ion mobility coefficients (1/K0). To perform diaPASEF, we used py_diAID , a python package, to assess the precursor distribution in the m/zion mobility plane to generate a diaPASEF acquisition scheme with variable window isolation widths that are aligned to the precursor density in m/z. Data was acquired using twenty cycles with three mobility window scans each (creating 60 windows) covering the diagonal scan line for doubly and triply charged precursors, with singly charged precursors able to be excluded by their position in the m/z-ion mobility plane. These precursor isolation windows were defined between 350 -1250 m/z and 1/k0 of 0.6 -1.45 V×s/cm 2 .