PRM analysis of phosphopeptides of Nab2, Foxk1, HURP, ERK1/2, NPM, and Hspa4 from ΔB-Raf:ER cells.

ΔB-Raf:ER cells were treated with U0126 or 4-HT for 30 min in four biological replicates. Total proteins from cell lysates were digested with Typsin/Lys-C mix, and phosphopeptides were enriched by IMAC using Fe-NTA columns. After data-dependent LC-MS/MS analysis, phosphopeptides of Nab2, Foxk1, HURP, ERK1/2, NPM, and Hspa4 were selected and quantified by targeted MS using the PRM method. Targeted MS/MS scans were acquired by a time-scheduled inclusion list at a resolution of 70,000, an AGC target of 2e5, an isolation window of 2.0 m/z, a maximum injection time of 500 ms, and a normalized collision energy of 27. Time alignment and relative quantification of the transitions were performed using Skyline software.