Proteome profiling of HepG2 cell line

We processed 1 mln cells, diluted in 150 μL of 50 mM HEPES buffer. After three freeze-thaw cycles and centrifugation (4 °C, 15,000 rpm, 15 min), 135 μL of supernatant was collected and treated with 15 μL of the cocktail comprised from 1 mg of RapiGest™ SF Surfactant, 60 μL 0.5 M CAA, 20 μL 0.5 M tris(2-carboxyethyl)phosphine hydrochloride (TCEP), buffered by 20 μL 1 M HEPES. The reaction lasted for 30 min at 70 °C with vigorous shaking at 1500 rpm in darkness.

Enzymatic digestion was performed with trypsin, added to reduced and alkylated proteins to achieve the estimated ratio to the substrate as 1:40 (w/w). The trypsinolysis was incubated for 4 h at 37 °C. The digestion was quenched by trifluoroacetic acid (to pH < 3 finally), incubated for 30 min at 37 °C, and centrifuged at 13,000 g for 10 min. The supernatant (200 uL) with peptides was collected and transferred into the EvoTip to proceed HPLC-MS/MS analysis.

EvoSep One chromatography system connected to Orbitrap Fusion Tribrid mass spectrometer was used to separate and identify the peptides.

Mass spectra were recorded in the positive ion mode. Data were obtained using the Orbitrap Exactive analyzer with a resolution of 70,000 (at m/z 400) for MS and 15,000 (m/z 400) for MS/MS scans. Higher energy collisional dissociation (HCD) was used to fragment the peptides. The signal threshold was established at 17,500 for an isolation window of 1 m/z. The first mass of HCD spectra was set to 100 m/z. The collision energy was set to 35%. Fragmented predecessors were dynamically excluded from targeting for 10 s. In addition, singly charged ions and ions with no defined charge conditions were excluded from triggering MS/MS scans. Three HPLC-MS/MS repetitions were accomplished for each sample.

Raw proteome data were transformed into mgf files by MSConvert (v. 3). To process proteome data we used SearchGUI software (v. 4.1.24) against SwissProt library of human canonical and alternatively spliced protein sequences. The false discovery rate (FDR) cut-off for peptide-spectra matches, peptides, and proteins was ≤1%. We used the following search parameters: enzyme specificity – trypsin; maximum one missed cleavage; carbamidomethylation of cysteine regarded as fixed modification; oxidation of methionine as variable modifications; precursor mass tolerance of 10 ppm; product mass tolerance of 0.05 Da.