S were then incubated with trypsin in a trypsin/protein ratio of 1:50 (w/w) at 37 overnight. They were further desalted and concentrated using an Oasis HLB sample cartridge column (Waters Corporation). Finally, the purified peptides were dried at 4 by vacuum freeze-drying. Two independent samples were prepared and processed (biological replicates) for each test.2DLC S/MS analysisIt was transferred into original fermentation medium (pH 8.0, CK) and 10? mol/L Cu2+ added fermentation medium (pH 8.0, Cu) respectively. In the lag phase (0? h), logarithmic phase (4?8 h), and early stage of the stationary phase (18?8 h), samples were taken at intervals of every 2 h, and after proper dilution (diluted 30-fold in this study), calculated cell using a hemocytometer analysis, under a microscope, pH measurements. Glucose concentration in medium was measured using 3,5-dinitrosalicylic acid method as Yang et al. previously described [34]. After 28 h of cultivation, B. thuringiensis X022 in CK but remained in the stationary phase for another 8 h in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/15501003 Cu were observed from this time onward.Total bacterial proteins extraction, trypsin digestionThe four trypsin digested samples (two replicates) were separated by 2D-HPLC equipped with a strong cationexchange column (BioBasic SCX; 0.32 mm ? 100 mm, 5 m) and a reversed-phase column (BioBasic-C18; 0.1 mm ?150 mm, 5 m), and then analyzed by MS/MS using an LTQ XL mass spectrometer (ThermoFisher, San Jose, CA, USA) equipped with a homemade nano-ionization source. The procedures were carried out as Huang et al. clearly described previously [10].Database search and functional classificationB. thuringiensis X022 cultivated in original fermentation medium and 10-6 mol/L Cu2+ added fermentation medium were harvested from three replicates and mixed together at Gemcitabine (hydrochloride) the time point of 44 h cultivation. The procedure of proteins extraction and trypsin digestion was carried out as previously described [34, 35]. Briefly, after washed with PBS buffer (10 mM, pH 7.8), The cells were mechanically disrupted with disposable grinding pestles, and then treated with ultrasonication (SCIENTZ 98-III) for 10 min at 4 . After incubated at 4 for 30 min, the mixtures were centrifugation with speed of 12,000g at 4 for 30 min. The supernatant of proteins was quantified using 2D Quant kit (Amersham Biosciences, Piscataway, NJ, USA). The proteins extracted for 2D-LC S/ MS analysis were tested with SDS-PAGE (AdditionalAn in-house database was constructed with the protein sequences downloaded from the Uniprot Knowledgebase (Swiss-Prot plus TrEMBL) protein database (http:// www.uniprot.org) as a FASTA-formatted sequence that included all B. thuringiensis subspecies. And the search results were further validated manually and classified into functional categories according to their annotated functions in the Uniprot Knowledgebase as well as to homology/functions according to the BioCyc (http:// www.biocyc.org/) and KEGG (http://www.genome.ad.jp/ kegg/kegg2.html) metabolic pathway databases [11].Semiquantification analysis of proteins expressionProtein abundance was determined by semiquantitative analysis as described earlier [36, 37]. Briefly, each protein abundance was obtained from emPAI value. The emPAI semi-quantitative of ICPs, in Additional file 1: Table S3, which calculated as emPAI = 10SC/OP - 1. SC is the total spectral count of MS/MS spectra for each detected protein. The OP was obtained after in silico trypsinization o.