Nanoelectrospray capillaries were made from borosilicate glass (OD 1 mm, ID 0.78 mm, 10 cm length, Sutter Instrument, item # BF100-78-10) using a micropipette puller (Sutter Instrument, model P-1000). The puller program was set to heat 495, pull 5, velocity 40, delay 235, pressure 200, and ramp 496. The resulting capillaries had openings of ~ 1 µm. Ten µL protein solution (~ 17 mg/mL) was buffer exchanged into 100 mM ammonium acetate (pH ~ 6.7) using a size exclusion spin column (Zeba Micro Spin Desalting Columns, 7 kDa MWCO, 75 µL) following manufacturer’s protocol. The resulting solution had a concentration of ~ 13 mg/mL as estimated by Nanodrop A280 measurement. Mass spectra were collected on an ion mobility–mass spectrometer (Waters Synapt G2s-i). The solution was further diluted to a desirable concentration and loaded into the glass capillary, where a platinum wire was inserted to apply a voltage of 500 V to sustain a nano-electrospray. Source temperature was set to 303 K and the sampling cone to 50 V to minimize protein activation. Gas flows were set to 3 mL/min for trap, 120 mL/min for helium cell, and 60 mL/min for ion mobility. Bias voltages were set to 45 V for trap, 35 V for helium cell, and 3 V for IMS. Traveling waves were set to 150 m/s 3 V for trap, 500 m/s 20 V for ion mobility, and 70 m/s 3 V for transfer. Ion mobility mass spectra were average over 1 min. Data were visualized in Driftscope and annotated manually.

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