The Synapt G2 HDMS was coupled to the automated chip-based nanoESI device (TriVersa NanoMate, Advion, Ithaca, NY, USA). The cone voltage of the Synapt G2 was fixed to 80 V to avoid in-source ion activation while ensuring ion transmission. The backing pressure was 6 mbar. The argon flow rate was set to 5 mL/min. Ions were focused in the helium cell (120 mL/min), prior to IM separation. The N2 flow rate in the IM cell was 60 mL/min. The wave height and velocity were fixed to 40 V and 850 m/s, respectively. Drift times were converted into CCS values using avidin (for middle-up level data), concanavalin A, alcohol dehydrogenase and pyruvate kinase (for intact-level data) as external calibrants [53,54]. ATDs were extracted using MassLynx v4.1.

CIU experiments were carried out by increasing the collision voltage in the trap cell from 0 to 200 V using steps of 5 V. CIU data were processed using the CIUSuite 2 v2.2 software [55]. ATDs were smoothed using a Savitsky-Golay algorithm with a window length of 5 and a polynomial order of 2. CIU acquisitions were performed in triplicate to generate averaged CIU fingerprints with their associated RMSD using the ‘Basic Analysis’ module of the CIUSuite 2 software. RMSDs under 15% between technical replicates account for a good reproducibility of CIU data (Table S2). CIU50 values, which allow to quantitatively assess unfolding transitions, were determined with the ‘Stability Analysis’ module.

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