Cell pellets (HCT116, HCT116 p53−/− and HT29) were lysed with a lysis buffer (8 M urea, 1 mM sodium orthovanadate, 20 mM HEPES, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, pH 8.0, 20 min, 4 °C) followed by sonication at 40% amplification by using a microtip sonicator (QSonica, LLC, Model no. Q55) and cleared by centrifugation (14 000 × g, 15 min, 15°C). Protein concentration was measured (Pierce BCA Protein Assay, Thermo Fisher Scientific, IL, USA) and a total of 100 µg of protein per sample was subjected for trypsin digestion. Typtic peptides were desalted using C18 Sep-Pak plus cartridges (Waters, Milford, MA) and were lyophilized for 48 h to dryness. The dried eluted peptides were reconstituted in buffer A (0.1 M acetic acid) at a concentration of 1 µg/µL and 5 µL was injected for each analysis.

The LC-MS/MS was performed on a fully automated proteomic technology platform [27]. that includes an Agilent 1200 Series Quaternary HPLC system (Agilent Technologies, Santa Clara, CA) connected to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The LC-MS/MS set up was used as described earlier (Ahsan et al., 2017, J Proteomics 2017, 165: 69–74). Briefly, the peptides were separated through a linear reversed-phase 90 min gradient from 0% to 40% buffer B (0.1 M acetic acid in acetonitrile) at a flow rate of 3 µl/min through a 3 µm 20 cm C18 column (OD/ID 360/75, Tip 8 µm, New objectives, Woburn, MA) for a total of 90 min run time. The electrospray voltage of 2.0 kV was applied in a split-flow configuration, and spectra were collected using a top-9 data-dependent method. Survey full-scan MS spectra (m/z 400–1800) were acquired at a resolution of 70,000 with an AGC target value of 3 × 106 ions or a maximum ion injection time of 200 ms. The peptide fragmentation was performed via higher-energy collision dissociation with the energy set at 28 normalized collision energy. The MS/MS spectra were acquired at a resolution of 17,500, with a targeted value of 2 × 104 ions or maximum integration time of 200 ms. The ion selection abundance threshold was set at 8.0 × 102 with charge state exclusion of unassigned and z =1, or 6 to 8 ions and dynamic exclusion time of 30 s.

Peptide spectrum matching of MS/MS spectra of each file was searched against the human database (UniProt) using the Sequest algorithm within Proteome Discoverer v 2.3 software (Thermo Fisher Scientific, San Jose, CA). The Sequest database search was performed with the following parameters: trypsin enzyme cleavage specificity, 2 possible missed cleavages, 10 ppm mass tolerance for precursor ions, 0.02 Da mass tolerance for fragment ions. Search parameters permitted variable modification of methionine oxidation (+15.9949 Da) and static modification of carbamidomethylation (+57.0215 Da) on cysteine. Peptide assignments from the database search were filtered down to a 1% FDR. The relative label-free quantitative and comparative among the samples were performed using the Minora algorithm and the adjoining bioinformatics tools of the Proteome Discoverer 2.3 software. To select proteins that show a statistically significant change in abundance between 2 groups, a threshold of 1.5-fold change with P-value (0.05) were selected.

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