2.10 Proteomics sequencing analysis
Quantitative proteomics analysis was performed by PTM BioLab, Inc. (Hangzhou, China) as a customer service. Approximately 0.1 g of each sample was pulverized in liquid N and mixed with four volumes of acetone containing 10% trichloroacetic acid. The samples were stored at- 20 ℃ for 4 h. The supernatant was discarded and the precipitate was washed three times with ice-cold acetone. The precipitate was air-dried and extracted with SDS/phenol method. Following trypsinization, peptides were reconstituted in 0.5 M TEAB and labeled with a TMTsixplex™ kit (ThermoFisher Scientifc).
The peptides were fractionated and the resulting peptides were subjected to an NSI source for analyzed by tandem mass spectrometry (MS/MS) in Q ExactiveTM (Thermo Scientific, US) coupled online to the UPLC-MS/MS. The acquired data were analyzed using the MaxQuant search engine (v.1.5.2.8). Proteins with a fold change (FC) of >1.2 or <0.833, plus a P -value <0.05, were set as thresholds for significant up- and down- regulation, respectively. The differentially expressed proteins (DEPs) were annotated using sub-cellular localization predictions (WolF Psort), Clusters of Orthologous Groups (COG/KOG) (https://www.ncbi.nlm.nih.gov/research/cog-project/), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways (http://www.genome.jp/kegg/kaas/) (Galperin et al. , 2021; Horton et al. , 2007; Moriya et al. , 2007).