*2.3. HRMS Analysis*

Operating conditions for the HESI source used in a positive ionization mode were: +3.5 kV spray voltage, 320 ◦C capillary and probe heater temperature, sheath gas flow rate 36 a.u., auxiliary gas flow rate 11 a.u., spare gas flow rate 1 a.u. (a.u. refer to arbitrary values set by the Exactive Tune software) and S-Lens RF level 50.00. Nitrogen was used for sample nebulization and collision gas in the HCD cell. The aliquots of 1 μL of the solutions of the samples (ca. 20 μg mL<sup>−</sup>1) were introduced into the mass spectrometer through the UHPLC system. Each chromatographic run was carried out isocratically with a mobile phase consisting of water-acetonitrile-methanol-acetic acid (25:50:25:0.2). The solvent flow rate was 300 μL min<sup>−</sup>1. Full MS—ddMS2 (Top5) was used as an MS experiment, where in full scan MS, the resolution, automatic gain control (AGC) target, maximum injection time (IT), and mass range were 70,000 (at *m/z* 200), 3 × 106, 100 ms, and *m/z* 100–500, respectively. The instrument parameters for ddMS<sup>2</sup> scans were as follows: the resolution was 17,500 (at *m/z* 200), AGC target was 1 × 105, maximum IT was 50 ms, loop count was 5, isolation window 2.0 *m/z*, stepped normalized collision energy (NCE) was set to 10, 20, 60. The data-depended (dd) settings were as follows: maximum ACG target was 5 × 104, dynamic exclusion was set to 1 s, preferred peptide match and switched on isotope exclusion were used. Xcalibur (Thermo Fisher Scientific, Waltham, MA, USA) ver. 4.0 was used for data acquisition and processing.
