JSH-23 protein concentrations were determined using Bradford protein assay (Bio-Rad) according to the manufacturer’s instructions. 2-DE Protein extracts (150 μg) were loaded onto 17-cm strips with a pH range of 4 to 7 (Bio-Rad), focused for 60,000 V.h, and then separated on a 12% SDS-polyacrylamide gel as reported previously [12]. The gels were stained with Bio-Safe Coomassie (Bio-Rad) and scanned on a GS-800 Calibrated Densitometer (Bio-Rad). Image analysis Image analysis of the 2-DE gels was performed using the PD Quest PRN1371 8.0.1 software (Bio-Rad).
Three gels were produced from independent cultures of each strain and only spots that were present on the three gels were selected Savolitinib purchase for inter-strain comparison. Spot intensities were normalized to the sum of intensities of all valid spots in one gel. For analysis of changes in protein expression during bile salt
exposure, a protein was considered to be under- or overproduced when changes in normalized spot intensities were of least 1.5-fold at a significance level of p < 0.05 (Student's t test for paired samples), as previously described [14]. Regarding proteome comparison between strains, proteins were considered differentially produced when spot intensities passed the threshold of a twofold difference (one-way ANOVA, p-value < 0.05), as described previously [12]. LC-MS analysis Spots of interest were subjected to tryptic in-gel digestion and analyzed by chip-liquid chromatography-quadrupole time of
flight (chip-LC-QTOF) using an Agilent G6510A QTOF mass spectrometer equipped with an Agilent 1200 Nano LC system and an Agilent HPLC Chip Cube, G4240A (Agilent Technologies, Santa Clara, CA, USA), as described previously [12]. Briefly, one microliter of sample was injected using an injection loop of 8 μL, a loading flow rate of 3 μL/min for 4 min and a solvent made of ultra-pure water and acetonitrile (HPLC-S gradient grade, Biosolve, Valkenswaard, The Netherlands) (97/3 v/v) with 0.1% formic acid (98-100%, Merck). For the analytical elution, a 24 min gradient from 3 to 60% of acetonitrile in ultra-pure water with 0.1% formic acid was applied at a flow rate of 300 nL/min. ESI in positive mode with 1850 capillary voltage was used. The data were collected in centroid Smoothened mode using extended dynamic range at mass range of m/z 200-2000 both in MS1 and MS/MS and using two method with different scanning speed: one slow with a scan rate of 1 spectra/s for both MS1 and MS/MS, and one fast scan rate of 0.25 spectra/s for both MS1 and MS/MS. For data acquisition and data export, MassHunter version B.02.0.197.0 (Agilent Technologies) was used. Protein identification After data acquisition, files were uploaded to the in-house installed version of Phenyx (Geneva Bioinformatics, Geneva, Switzerland) for searching the NCBInr (r.