3.3. Mass Spectrometry

All Electrospray ionization-mass spectrometry (ESI-MS) experiments were performed on the LCMS-9030 qTOF Shimadzu (Shimadzu, Kyoto, Japan) device, equipped with a standard ESI source and the Nexera X2 system. Analysis was performed in the positive ion mode between 100 and 3000 m/z. LCMS-9030 parameters: nebulizing gas—nitrogen, nebulizing gas flow 3.0 L/min, drying gas flow—10 L/min, heating gas flow—10 L/min, interface temperature 300 °C, desolvation line temperature—400 °C, detector voltage—2.02 kV, interface voltage 4.0 kV, collision gas—argon, mobile phase (A) H₂O + 0.1% HCOOH, (B) MeCN + 0.1% HCOOH, mobile phase total flow—0.3 mL/min. The injection volume was optimized depending on the intensity of the signals observed on the mass spectrum within the range of 0.1 to 1 μL. All obtained signals had a mass accuracy error in the range of 1 ppm. The concentration of peptide was 0.1 mM, and M:L molar ratio was 1:1. Samples were prepared in a mixture of water/methanol (50/50 v/v) at pH 7.40. All of the used solvents were of LCMS grade. The obtained data were analyzed by ACD/Spectrus Processor 220.2.0 (ACD/Labs, Toronto, ON, Canada).