*4.1. Preparation of Myoglobin-Containing Solution*

To demonstrate the procedure, electrospray-compatible solutions of myoglobin with neutral pH, in which binding activities are maintained for performing the ITEM-TWO experiments, are prepared. A stock solution was first prepared by dissolving 5.22 mg of myoglobin (lot # 60K7007, Sigma-Aldrich, Steinheim, Germany) in 1 mL of 200 mM ammonium acetate. Next, 50 µL of the stock solution was diluted with 450 µL of 200 mM ammonium acetate. The concentration of the resulting solution was determined to be 0.5 µg/µL using a QubitTM 2.0 Fluorometer (Carlsbad, California, USA). Finally, 100 µL of the 0.5 µg/µL myoglobin solution were further diluted to 200 µL with 200 mM ammonium acetate buffer, pH 7, to obtain a final concentration of 0.25 µg/µL. For each measurement, ca. 3 µL of the 0.25 µg/µL myoglobin solution were loaded into nanoESI capillaries using a microloader pipette tip (Eppendorf, Hamburg, Germany) and were electrosprayed directly.
