Proteomic Analysis of Gemcitabine and DTLL

The proteomic analysis of the cell samples was conducted using the combination of DIA and a data-dependent acquisition (DDA)-based ion library. After treatment with 2 μM gemcitabine, 0.1 nM DTLL and both at 37 °C for 4 h, cells were harvested and lysed with lysis buffer (8 M urea, 100 mM Tris-HCl pH 7.6, 1 mM PMSF), and then centrifuged at 18,000 × g for 15 min at 4 °C. The extracted protein in the supernatant was quantified using a BCA protein assay kit (Beyotime Biotechnology, China) and digested in trypsin (Promega, Madison, WI) after reduction and alkylation using the FASP (filter aided sample preparation) method. The concentration of digested peptides was determined by measuring the absorbance at 280 nm using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). An EASY-nLC 1200 chromatography system and an Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) were used for mass spectrometry acquisition and analysis. The DIA analysis was performed according to a previous study^{71 (link)}. All results were filtered by a Q-value cutoff of 0.01 (corresponding to an FDR of 1%). The P-value estimator was performed by the Kermel Density Estimator. The area was used for protein quantification. Every peptide was validated with at least three fragments.