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).