2.12. Shotgun Proteomic Analysis of Antibacterial Protein Components in Z. morio Hemolymph

A shotgun proteomic analysis was performed on the uninduced Z. morio hemolymph, on the antibacterial protein solution after ultrafiltration, and the antibacterial protein components of Z. morio hemolymph, which was purified by a molecular sieve mentioned in 2.11. The protein cleavage was used by SDT (4% SDS, 100 mM Tris-HCl, 1 mM dithiothreitol (DTT), pH 7.6), and the quantification was measured by Beyotime Biotechnology BCA kit (Shanghai, China). The protein sample was digested with filter-aided sample preparation (FASP), and the peptide was desalted by C18 Cartridge (66872-U Sigma, Shanghai, China. After lyophilizing the peptide, 40 μL 0.1% formic acid solution (FA, 06450 Fluka, Charlotte, NC, USA) was added to redissolve, and the peptide was quantified. Each sample was separated by the HPLC system Easy nLC (Thermo Scientific, Waltham, MA, USA) and analyzed by a Q-Exactive mass spectrometer. The MaxQuant software (version 1.5.3.17, Max Planck Institute of Biochemistry, Martinsried, Germany) was used for database mass spectrometry and quantitative analysis.