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).