Protein alkylation and digestion, and mass spectrometry analysis

Samples of V. destructor OBP1, OBP2 and OBP5 (20–50 μg) dissolved in 0.1 M tetraethylammonium bicarbonate (TEAB), pH 6.5, containing 4 M guanidinium chloride, were treated with iodoacetamide (0.5 M final concentration) for 30 min, in the dark; then, proteins were insolubilized by addition of 6 vol of cold acetone, leaving the samples at -20 °C, overnight. After centrifugation at 12,000 rpm at 4° C, for 20 min, supernatants were removed, and recovered pellets were dried with a SpeedVac device (ThermoFisher Scientific, USA). Recovered proteins were dissolved in 0.05 M TEAB, pH 6.5 (2 µg/μL final concentration), treated with trypsin (1:10 w/w enzyme/substrate) for 16 h, at 37 °C, then with chymotrypsin (1:8 w/w enzyme/substrate) for 16 h, at 37 °C. Protein digests were desalted with ZipTip C18 (Millipore, USA) and directly analyzed with a UltiMate 3000 HPLC RSLC nano-chromatographer (ThermoFisher Scientific) linked on-line through a nano-Spray ion source (Thermo Fisher Scientific) to a Q-ExactivePlus mass spectrometer (Thermo Fisher Scientific)³³. Peptides were separated on an Acclaim PepMap RSLC C18 column (150 mm × 75 μm ID; 2 μm particle size; 100 Å pore size) (Thermo Fisher Scientific), at a flow rate of 300 nL/min, using a gradient of solvent B (19.92/80/0.08 v/v/v water/acetonitrile/formic acid) in solvent A (99.9/0.1 v/v water/formic acid). Solvent B started at 3%, increased linearly to 40% in 45 min, then raised to 80% in 5 min, where it remained for additional 4 min, before rapidly returning to 3%. Mass spectrometer worked in positive polarity using a data-dependent mode, performing a full MS1 scan in the range m/z 345–1350, at a nominal resolution of 70,000, followed by MS/MS scans of the 10 most abundant ions in high energy collisional dissociation mode³⁴. MS/MS spectra were acquired in a dynamic m/z range, with a nominal resolution of 17,500, a normalized collision energy of 28%, an automatic gain control target of 50,000, a maximum ion injection time of 110 ms, an isolation window of 1.2 m/z and a dynamic exclusion setting of 20 s.