Label-free Quantitative Proteomics of HeLa Cells

Label-free quantitative proteomic analysis was carried out as previously described [34 (link),35 (link)]. Briefly, HeLa cells (1 × 10⁶) were harvested, washed twice in PBS, and lysed in buffer containing 6M GnHCl, 20 mM tris(2-carboxyethyl)phosphine (TCEP; Pierce^TM, Thermo Fisher Scientific, Waltham, MA, USA), 40 mM 2-chloroacetamide (CAA; Sigma-Aldrich) in 100 mM Tris, pH 8.0. The lysate was heated to 95 °C for 2 min and then sonicated in a Bioruptor sonicator (Diagenode) at the maximum power setting for 10 cycles of 30 s each. The entire process of heating and sonication was repeated once, and then the sample was diluted 10-fold with digestion buffer (25 mM Tris, pH 8, with 10% acetonitrile). Protein extracts were digested for 4 h with endoproteinase lysC, followed by the addition of trypsin for overnight digestion. After digestion, peptides were purified and loaded for mass spectrometric analysis. Raw data were processed using the MaxQuant computational platform [46 (link)]. The peak list was searched against Human Uniprot databases, with an initial precursor and fragment tolerance of 4.5 ppm. The match between the run feature was enabled, and proteins were quantified across samples using the label-free quantification algorithm in MaxQuant as label-free quantification (LFQ) intensities [47 (link)].