Protein Identification and Quantification Protocol

Protein samples were added to four volumes of acetone. The protein precipitate was preserved and then dissolved in 8 M urea buffer (pH 8.5, 100 mM Tris–HCl). Then, 10 mM trichloroethyl phosphate and 20 mM iodoacetamide were reacted with the protein precipitate for half an hour to allow denaturation and alkylation. Subsequently, the urea concentration in the sample was diluted to 1 M and subjected to enzymatic hydrolysis. These enzymatic fragments were separated and desalted by 2D-HPLC equipped with a strong cation-exchange column (BioBasic SCX; 0.32 mm × 100 mm, 5 μm) and a reversed-phase column (BioBasic-C18; 0.1 mm × 150 mm, 5 μm) and then analyzed by LC–MS/MS using the Thermo Q-Exactive Plus (ThermoFisher, San Jose, CA, USA) equipped with an ultra-high performance liquid chromatography unit (Thermo Scientific Dionex Ultimate 3000) and a Nanospray Flex Ion-Source (Thermo Scientific) [16 (link), 20 (link), 28 –30 (link)]. After the original mass spectrometry data were converted into the mgf format file by the corresponding tools, the Maxquant software (according to the Bt protein library) was used to search for the identification and quantitative information extraction of the corresponding database. Significantly different proteins were screened using metaX software. Finally, conduct the GO, KEGG Pathway, eggNOG bioinformatics analysis.