Hela cells were grown over 8 passages in High Glucose DMEM [12.43 g/l Dulbecco’s Modified Eagle’s Medium (Caisson Laboratories Inc., North Logan, UT, USA), 4.5 g/l D-(+)-Glucose anhydrous (Fluka, Buchs, Switzerland), 30 mg/l Glycine (Fluka)] supplemented with either light (Sigma-Aldrich) or heavy (13C 15N, Sigma-Aldrich) isotope-labeled lysine and arginine at 37°C and 5% CO2.
Cells were harvested at 80% confluency by trypsinization, washed three times with ice cold PBS (GIBCO (Invitrogen), Paisley, UK) and the cell number was determined using a Neubauer chamber. Hela cells were spun down at 300 ×g and resuspended in one cell pellet volume PBS. Two pellet volumes of 8 M Urea (Sigma-Aldrich, Buchs, Switzerland) containing 50 mM ammonium bicarbonate (Sigma-Aldrich) and 0.1% RapiGest (Waters, Baden, Switzerland) were thoroughly mixed with the resuspended cells. Subsequent to sonication (80% amplitude, 0.6 cycle, 1 min) cell debris was spun down at 16000×g.
The protein concentration of the lysate was measured by BCA assay (bicinchoninic acid, Thermo Scientific, Reinach, Switzerland). Proteins were reduced with 5 mM TCEP (tris(2-carboxyethyl)phosphine, Thermo Scientific) at 37°C for 15 min and alkylated with 10 mM iodoacetamide (Sigma-Aldrich) for 30 min in the dark. Proteins were first digested with lysyl endopeptidase (Wako Chemicals, Neuss, Germany) at an enzyme – substrate ratio of 1 to 50 (w/w) at 35°C for 2 hours. After dilution with 50 mM ammonium bicarbonate to 0.8M urea trypsin (Promega) was added at the same ratio. Tryptic digestion was carried out overnight at 37 °C. Peptides were acidified with 1% trifluoroacetic acid (TFA, Thermo Scientific) and purified by solid-phase extraction using C18 cartridges (Sep-Pak, Waters). The SPE eluate was evaporated to dryness and reconstituted in 3% acetonitrile (Thermo Scientific) and 0.2% formic acid (Sigma-Aldrich).
Cells were harvested at 80% confluency by trypsinization, washed three times with ice cold PBS (GIBCO (Invitrogen), Paisley, UK) and the cell number was determined using a Neubauer chamber. Hela cells were spun down at 300 ×g and resuspended in one cell pellet volume PBS. Two pellet volumes of 8 M Urea (Sigma-Aldrich, Buchs, Switzerland) containing 50 mM ammonium bicarbonate (Sigma-Aldrich) and 0.1% RapiGest (Waters, Baden, Switzerland) were thoroughly mixed with the resuspended cells. Subsequent to sonication (80% amplitude, 0.6 cycle, 1 min) cell debris was spun down at 16000×g.
The protein concentration of the lysate was measured by BCA assay (bicinchoninic acid, Thermo Scientific, Reinach, Switzerland). Proteins were reduced with 5 mM TCEP (tris(2-carboxyethyl)phosphine, Thermo Scientific) at 37°C for 15 min and alkylated with 10 mM iodoacetamide (Sigma-Aldrich) for 30 min in the dark. Proteins were first digested with lysyl endopeptidase (Wako Chemicals, Neuss, Germany) at an enzyme – substrate ratio of 1 to 50 (w/w) at 35°C for 2 hours. After dilution with 50 mM ammonium bicarbonate to 0.8M urea trypsin (Promega) was added at the same ratio. Tryptic digestion was carried out overnight at 37 °C. Peptides were acidified with 1% trifluoroacetic acid (TFA, Thermo Scientific) and purified by solid-phase extraction using C18 cartridges (Sep-Pak, Waters). The SPE eluate was evaporated to dryness and reconstituted in 3% acetonitrile (Thermo Scientific) and 0.2% formic acid (Sigma-Aldrich).
