Easy nlc 1200 nano lc system

LC–MS/MS was used to confirm covalent adduction and site identification for two positive hits generated from the Nedd4 inhibition screen that demonstrated low IC₅₀ values. 30 µg of N-terminal His-tagged and C-terminal biotinylated Nedd4 (residue 1–900) was mixed with 200 µM of compound 25 or 81 (Enamine Cat. No. Z65886524 and Z1205657985) solubilized in 1% DMSO and incubated at RT for 3 h. Samples were run on SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific cat no. 20278). Gels were destained in water until the desired contrast was reached. The Nedd4 bands were cut and stored in 1% acetic acid at 4 °C until use. Samples were submitted to the SPARC Molecular Analysis Centre at the Hospital for Sick Children (Toronto, ON, CA) for LC–MS/MS analysis. Each gel band was cut into 3 pieces and digested with 0.8 µg pepsin, 0.6 µg trypsin and 0.6 µg chymotrypsin separately. The peptides obtained through the proteolytic digest were combined then lyophilized and resuspended in 0.1% formic acid. These samples were then subjected to MS/MS spectroscopy by an EASY-nLC 1200 nano-LC system + Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. MS data was analyzed using Scaffold PTM 4.0.1.

A Q-Exactive HF mass spectrometer equipped with an EASY-nLC 1200 nano-LC system (Thermo Fisher Scientific, United States) was used for LC–MS analysis. The samples were dissolved in 0.1% FA and loaded into a 15 cm length reversed-phase column (150 nm id) packed with Ultimate XB-C18 1.9 μm resin (Welch materials). The constant flow rate was 600 nL/min. The eluted peptides were analyzed by data-dependent MS2 acquisition (DDA). Higher-energy collision dissociation (HCD) with a normalized collision energy of 27% was used for peptide fragmentation.