Sample preparation for proteomics analysis
Cells (5 x 107) were harvested on day 6 and 10 of the culture, quenched using a -20°C ethanol bath and kept cold in a CoolRack® (Corning) during the sample preparation. Cells were spun down at 1000g, 1min, -5°C. The cell pellets were washed two times with cold 0.9% NaCl solution. Proteins were extracted in 1mL of 6M guanidine solution, boiled 5min at 95°C, and vortexed. Samples were then spun down 10 min at 17000g and supernatants were stored at -80°C. Protein concentration was determined in samples diluted 10 times following the protocol of the PierceTM BCA Protein assay (Thermo Scientific). 50µg of each samples was reduced and alkylated using tris(2-carboxyethyl)phosphine and chloroacetamide at a final concentration of 10mM and 40mM respectively. Samples were then digested using LysC (1:50 protein ratio, incubation 3-4h at 37°C) and Trypsin (1:10 protein ratio, incubation overnight at 37°C). Samples were then diluted in trifluoroacetic acid to a final concentration of 1% (v/v) to stop the digestion. Prior to labelling, the peptides were desalted using C18 StageTips. Samples and internal standard were labelled using the 11-plexes Tandem Mass Tag reagents (Thermo Scientific). Each Injection was fractionated using ion exchange column and analyzed on Q-Exactive mass spectrometer (Waters).