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.