2.5. Phosphopeptide enrichment, MS/MS analysis and relative quantitation of the phosphopeptides

K562 or K562‐STI‐R cells in a logarithmic growth phase were subjected to phosphopeptide enrichment and proteomic analysis as described previously (Chorzalska et al., 2017). Five independently revived and expanded cellular clones per cell line were harvested, lysed, and used to prepare tryptic digested peptides. The peptides were lyophilized, and phosphopeptide enrichment was performed using Titansphere Phos‐TiO tips (GL Sciences, Tokyo, Japan) following the manufacturer's protocol with minor modifications (Ahsan et al., 2017). LC‐MS/MS was performed on a fully automated proteomic technology platform (Yu and Salomon, 2010) that utilizes an Agilent 1200 Series Quaternary HPLC system (Agilent Technologies, Santa Clara, CA, USA) connected to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). MS/MS spectra were searched against a human‐specific database (UniProt; downloaded 2/1/2013) using mascot v. 2.4 (Matrix Science, Ltd, London, UK). Peptide assignments from the database search were filtered down to 1% false discovery rate (FDR) by a logistic spectral score, as previously described (Chorzalska et al., 2017; Elias and Gygi, 2007; Yu et al., 2009). To validate the position of phosphorylation sites, the Ascore algorithm (Beausoleil et al., 2006) was applied and the reported phosphorylation site position reflected the top Ascore prediction. Relative quantification of phosphopeptides abundance was performed via calculation of selected ion chromatograms (SIC) peak areas. Retention time alignment of individual replicate analyses was performed as previously described (Demirkan et al., 2011).