2.3 Protein extraction and sequencing
Total proteins were extracted from the fourth instar whole body of two insects with three biological replicates according to a previously described protocol (Unwin et al., 2010) with minor modifications. Samples were ground to a powder in liquid nitrogen and lysed with 2 mL lysis buffer containing 8 M urea, 2 M thiourea, 0.1 % 3-[(3-cholamidopropyl) dimethylammonio propanesulfonate (CHAPS) (Amresco Ltd., USA) and 1 × Protease Inhibitor Cocktail (Roche, USA). The lysis solution was centrifuged at 4 °C, 13,000 × g for 15 min to collect the supernatant in a new tube and then save it at -80 °C until use. The protein concentration was determined using a 2-D Quant Kit (GE Healthcare, USA), and quality was examined with SDS-PAGE (Beyotime, China). Protein digestion was conducted using trypsin (Promega, USA) at 37 °C overnight, and peptides were dried in a centrifugal vacuum concentrator.
According to a previously described protocol, protein isolation and labeling were performed using the 8-plex iTRAQ (Applied Biosystems) according to a previously described protocol (Wang et al., 2015) with some modifications. Sample peptides were subjected to nano-electrospray ionization, followed by tandem mass spectrometry (MS/MS) in an Orbitrap Q-Exactive plus system (Thermo Fisher Scientific, USA). MS scans were obtained from m/z 350-1,800, with 40 precursors selected for MS/MS from m/z 100-1,800 using a dynamic exclusion of 40 s for the selected ions. The collision-induced dissociation (CID) energy was automatically set as 32 %. The database search strategy-based peptide matching tolerance was controlled below 10 ppm and 0.05 Da to prevent the omission of proteins.