Quality Control

to monitor Q-Exactive HF-X performance

Sample preparation.

HeLa Kyoto cells were cultured on 24-cm square cell culture trays, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco/Invitrogen) plus 0.2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) (all from Sigma-Aldrich). Cells were harvested by scraping into their medium, washed with ice-cold phosphate-buffered saline (PBS), snap-frozen in liquid nitrogen, and stored at −80°C. Frozen cell pellets were thawed and then resuspended in one pellet volume of extract buffer [20 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 20 mM b-glycerophosphate, 10 mM NaF, 10% glycerol, 0.1% NP-40, 1 mM okadaic acid (Alexis/Enzo Life Sciences), 0.2 mM Na3VO4, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mix (leupeptin, pepstatin and chymostatin, 10 mg/ml each)]. Cells were lysed with a Dounce homogenizer, and the crude extract was clarified by centrifugation at 14,000 rpm in a microfuge for 15min.

Protein concentration was measured using bradford. The protein pellet was dissolved in 8M urea, 0.5M ammoniumbicarbonate to a final protein concentration of 2 ug/ul. Proteins were reduced with dithiothreitol (DTT) (1ug/20ug protein) and alkylated using iodoacetamide (5ug/20ug protein). Then the solution was diluted to a concentration of 6M urea. Proteins were digested using LysC (enzyme/protein ratio 1:50) for 2 hours at 30°C. Predigested protein solution was diluted to a urea concentration of 0.8M and digested using trypsin (Promega) (enzyme/protein ratio 1:30) overnight at 37°C.

25ng of digested protein lysate was injected to the mass spectrometer.

Liquid chromatography.

The nano HPLC system used was an UltiMate 3000 RSLC nano system (Thermo Fisher Scientific, Amsterdam, Netherlands) coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), equipped with a Proxeon nanospray source (Thermo Fisher Scientific, Odense, Denmark). Peptides were loaded onto a trap column (Thermo Fisher Scientific, Amsterdam, Netherlands, PepMap C18, 5 mm × 300 μm ID, 5 μm particles, 100 Å pore size) at a flow rate of 25 μL min-1 using 0.1% TFA as mobile phase. After 10 min, the trap column was switched in line with the analytical column (Thermo Fisher Scientific, Amsterdam, Netherlands, PepMap C18, 500 mm × 75 μm ID, 2 μm, 100 Å). Peptides were eluted using a flow rate of 230 nl min-1, and a binary 30 min gradient, respectively 75 min.

The gradient starts with the mobile phases: 98% A (water/formic acid, 99.9/0.1, v/v) and 2% B (water/acetonitrile/formic acid, 19.92/80/0.08, v/v/v), increases to 35%B over the next 30 min, followed by a gradient in 5 min to 90%B, stays there for 5 min and decreases in 2 min back to the gradient 98%A and 2%B for equilibration at 30°C.

MS data acquisition.

The Q Exactive HF-X mass spectrometer was operated in data-dependent mode, using a full scan (m/z range 380-1500, nominal resolution of 60,000, target value 1E6) followed by MS/MS scans of the 10 most abundant ions. MS/MS spectra were acquired using normalized collision energy of 28, isolation width of 1.0 m/z, resolution of 30.000 and the target value was set to 1E5. Precursor ions selected for fragmentation (exclude all charge states except 2 to 6) were put on a dynamic exclusion list for 20 s. Additionally, the minimum AGC target was set to 5E3 and intensity threshold was calculated to be 4.8E4. The peptide match feature was set to preferred and the exclude isotopes feature was enabled. Two methods with reduced isolation width of 0.7 and 0.4 m/z and otherwise identical settings were used to benchmark isolation performance of the quadrupole.

Data analysis.

Data was analysed in SimpatiQCo (Pichler et al). Therefore, SimpatiQCo performs a Mascot search (Perkins et al) (Matrix Science, London, UK; Version 2.2.07) against the human swissprot database. Oxidation of methionine and carbamidomethylation of cysteine were set as dynamic modifications. Trypsin was defined as the proteolytic enzyme. Mass tolerance was set to 10 ppm for precursors and 50 mmu for fragment masses. Spectra identified with a Mascot score below 25 were rejected.