Sequencing grade trypsin solution

Total protein was extracted from both IVM-resistant and susceptible lice (48-hours ten starved adult lice per sample, with four replicates of each). Samples were suspended in 200 μL of lysis buffer (8 M urea, 2 M thiourea, 100 mM NaCl, 25 Mm Tris, pH 8.2, complete protease inhibitor) and crushed with two 3-mm tungsten beads in TissueLyser at 25 Hz for four minutes (Qiagen, Courtaboeuf, France). After homogenization, the insoluble fractions were removed by centrifugation and soluble proteins were dialyzed twice using Slide-A-Lyzer MINI Dialysis Devices (Pierce Biotechnology, Rockford, USA) and dialysis buffer (50 mM ammonium bicarbonate, pH 7.4, 1 M Urea) following the manufacture’s protocol. Dialyzed fractions were collected, and protein concentration was determined by Bradford Protein Assay using Coomassie (Biorad, Marnes-la-Coquette, France). The dialyzed fractions were used as a template for global proteomic analysis. Briefly, 50 μg of total soluble proteins were reduced with 10 mM dithiothreitol for one hour at 30°C, alkylated with 20 mM iodoacetamide for one hour in the dark, and then digested by adding 2 μg of sequencing-grade trypsin solution (Promega, Charbonnières, France) for 20 hours at 37°C. The digested samples were then desalted using Pierce Detergent Removal Spin Columns (Thermo Fisher Scientific, Illkirch, France) following the manufacturer’s protocol.

In-gel digestion of protein spots from Coomassie Blue stained gels was carried out as previously described (Bahk et al. 2004 (link)). Briefly, selected protein spots were excised from stained gels and cut into pieces. The gel pieces were washed for 1 h at room temperature in 25 mM ammonium bicarbonate buffer, pH 7.8, containing 50% (v/v) acetonitrile (ACN). Following the dehydration of gel pieces in a centrifugal vacuum concentrator for 10 min, gel pieces were rehydrated in 50 ng of sequencing grade trypsin solution (Promega, Madison, USA). After incubation in 25 mM ammonium bicarbonate buffer, pH 7.8, at 37 °C overnight, the tryptic peptides were extracted with 5 µL of 0.5% formic acid including 50% (v/v) ACN for 40 min with mild sonication. The extracted solution was concentrated using a centrifugal vacuum concentrator. Before mass spectrometric analysis, the peptides solution was conducted to a desalting process using a reversed-phase column (Gobom et al. 1999 (link)). Briefly, after an equilibration step with 10 µL of 5% (v/v) formic acid, the peptides solution was loaded onto the column and washed with 10 µL of 5% (v/v) formic acid. The bound peptides were eluted with 5 µL of 70% ACN with 5% (v/v) formic acid.

Protein bands from the SDS-PAGE gels were excised and in-gel digested with trypsin (Promega, Madison, WI, USA). Briefly, protein bands were excised from the stained gels and cut into pieces. The gel pieces were washed for 1 h at RT in 25 mM ammonium bicarbonate buffer (pH 7.8) containing 50% (v/v) acetonitrile (ACN, Sigma-Aldrich). Following the dehydration of gel pieces in a centrifugal vacuum concentrator (Biotron, Korea) for 10 min, the gel pieces were rehydrated in sequencing-grade trypsin solution (Promega). After incubation in 25 mM ammonium bicarbonate buffer at 37 °C overnight, the tryptic peptides were extracted with 1% formic acid (FA, Sigma-Aldrich) containing 50% (v/v) ACN for 20 min with mild sonication. The extracted solution was concentrated using a centrifugal vacuum concentrator. Prior to mass spectrometric analysis, the peptide solution was subjected to a desalting process using a reversed-phase column.

The procedures for the in-gel digestion of protein spots excised from the Coomassie Blue-stained gels were performed as previously described^{48 (link)}. In brief, the protein spots were excised from the stained gel and cut into pieces. The gel pieces were washed for 1h at room temperature in 25 mM ammonium bicarbonate buffer, pH 7.8, containing 50% (v/v) acetonitrile (ACN). Following the dehydration of gel pieces in a centrifugal vacuum concentrator for 10 min, the gel pieces were rehydrated in 50 ng of sequencing grade trypsin solution (Promega, Madison, WI, USA). After incubation in 25 mM ammonium bicarbonate buffer, pH 7.8, at 37 °C overnight, the tryptic peptides were extracted with 5 uL of 0.5% formic acid containing 50% (v/v) ACN for 40 min with mild sonication. The extracted solution was concentrated using a centrifugal vacuum concentrator. Prior to mass spectrometric analyses, the peptide solution was subjected to a desalting process using a reversed-phase column^{49 (link)}. In brief, after an equilibration step with 10 μL of 5% (v/v) formic acid, the peptides solution was loaded on the column and washed with 10 μL of 5% (v/v) formic acid. The bound peptides were eluted with 5 μL of 70% ACN containing 5% (v/v) formic acid.

The experiment for in-gel digestion of protein spots from Coomassie Blue stained gels was performed as follows [8 (link),9 ]. Protein spots were resected from stained gels. The gel pieces were cleaned for 1hr at room temperature in pH 7.8, 25 mM ammonium bicarbonate buffer, containing 50% (v/v) acetonitrile (ACN). The gel pieces were dehydrated in a SpeedVac(Thermo fisher) for 10 min and were rehydrated in 10 μL (20 ng/μL) of sequencing grade trypsin solution (Promega, WI, USA). After gel pieces were incubated in pH 7.8, 25 mM ammonium bicarbonate buffer, at 37 °C overnight, the trypsin-digested peptides were extracted with 5 μL of 0.5% TFA containing 50% (v/v) ACN for 40 min with gentle sonication. The extracted peptide solution was reduced to 1 μL in a vacuum centrifuge.
After that, the peptides solution was subjected to a desalting process using a reversed-phase column [10 (link)]. A GEloader tip (Eppendorf, Hamburg, Germany) was packed with Poros 20 R2 resin (PerSpective Biosystems, MA, USA). After an equilibration step with 5% (v/v) formic acid, the peptides solution was loaded on the column and washed. The peptides were eluted with α-cyano-4-hydroxycinnamic acid (CHCA) and dropped onto MALDI plate (96 × 2; Applied Biosystems, Forster city, CA, USA).