Cells were transfected with ALK-targeting siRNA or control siRNA for 48 hours and treated with two different concentrations of the inhibitor lorlatinib or DMSO (30-min treatment) to enable comparison between conditions (fig. S7). Cells were washed in phosphate-buffered saline (PBS) and lysed for 10 min at 99°C in 6 M guanidine-HCl, 100 mM tris (pH 8.5), 5 mM tris(2-carboxyethyl)phosphine (TCEP), and 10 mM CAA, and whole-cell extracts were sonicated. Cell lysates were digested by Lys-C (Wako) in an enzyme/protein ratio of 1:100 (w/w) for 1 hour, followed by a dilution with 25 mM tris buffer (pH 8.5), to 2 M guanidine-HCl and further digested overnight with trypsin (Sigma-Aldrich; 1:100, w/w). Protease activity was quenched by acidification with TFA, and the resulting peptide mixture was concentrated on C18 Sep Pak Cartridges (Waters). Peptides were eluted with 40% ACN followed by 60% ACN. The combined eluate was reduced by SpeedVac, and the final peptide concentration was estimated by measuring absorbance at A280 on a NanoDrop (Thermo Fisher Scientific). Peptide (300 μg) from each sample was labeled with 1 of 11 different TMT reagents according to the manufacturer’s protocol (Thermo Fisher Scientific). After labeling, the samples were mixed and adjusted to 80% ACN and 6% TFA, and phosphopeptides were further enriched by two sequential rounds of titansphere chromatography as previously described (51). The eluted phosphopeptides were concentrated in a SpeedVac and fractionated with high-pH reversed-phase fractionation as described (74).

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