*4.2. Preparation of RNAse S-Containing Solution*

A stock solution of ca. 1 mg/mL was first prepared by dissolving the lyophilized powder (0.26 mg) of RNAse S (Lot # 52H7034, Sigma-Aldrich, Steinheim, Germany) in 0.26 mL of 200 mM ammonium acetate, pH 7.0. Then, 100 µL were transferred onto a Microcon centrifuge filter with a 3 kDa cutoff (Millipore Corp., Bedford, MA, USA) together with further 200 µL of 200 mM ammonium acetate solution. This solution was centrifuged for 30 min at 13,000 rpm and 23 ◦C. The eluate was discarded and 200 µL of 200 mM ammonium acetate solution were added onto the filter. This procedure was repeated for three times. Then, the filter was inverted, placed into a new tube, and centrifuged for 5 min at 4500 rpm at 23 ◦C. A resulting supernatant of approximately 800 µL was collected and the protein concentration was determined to be 0.78 µg/µL using a QubitTM 2.0 Fluorometer (Carlsbad, CA, USA) assay. For nano electrospray mass spectrometry 2.56 µL of the purified and concentrated RNAse S solution were diluted to a final concentration of 0.2 µg/µL with 7.44 µL of 10% methanol/200 mM ammonium acetate. For each measurement, ca. 3 µL of the RNAse S solution were loaded into nanoESI capillaries using a microloader pipette tip (Eppendorf, Hamburg, Germany).
