92 CHAPTER 4 peptide length was 7 amino acids and the minimum Andromeda score for modified peptides was 40, with the corresponding minimum delta score set at 1736. Proteins that could not be differentiated based on MS/MS spectra alone were grouped into protein groups (default MaxQuant settings). (Phospho)peptide identifications were propagated across samples using the match-between-runs option checked. Searches were performed with the label-free quantification option selected. A normalization factor derived from the total count of matched protein lysates was applied to scale peptide intensities for each pTyr capture. PROTEIN EXPRESSION PROFILING Protein lysates (50 μg) were separated on precast 4–12% gradient gels using the NuPAGE SDS‐PAGE system (Invitrogen, Carlsbad, CA). Following electrophoresis, gels were fixed in 50% ethanol/3% phosphoric acid solution and stained with Coomassie R‐250. Gel lanes were cut into five bands, and each band was cut into ~1 mm3 cubes. Gel cubes were washed with 50 mM ammonium bicarbonate/50% acetonitrile and were transferred to a 1.5 ml microcentrifuge tube, vortexed in 400 μl 50 mM ammonium bicarbonate for 10 min, and pelleted. The supernatant was removed, and the gel cubes were vortexed in 400 μl 50 mM ammonium bicarbonate/50% acetonitrile for 10 min. After pelleting and removal of the supernatant, this wash step was repeated. Subsequently, gel cubes were reduced in 50 mM ammonium bicarbonate supplemented with 10 mM DTT at 56°C for 1 h. The supernatant was removed, and gel cubes were alkylated in 50 mM ammonium bicarbonate supplemented with 50 mM iodoacetamide for 45 min at room temperature in the dark. Next, gel cubes were washed with 50 mM ammonium bicarbonate/50% acetonitrile dried in a vacuum centrifuge at 50°C for 10 min and covered with trypsin solution (6.25 ng/μl in 50 mM ammonium bicarbonate). Following rehydration with trypsin solution and removing excess trypsin, gel cubes were covered with 50 mM ammonium bicarbonate and incubated overnight at 25°C. Peptides were extracted from the gel cubes with 100 μl of 1% formic acid (once) and 100 μl of 5% formic acid/50% acetonitrile (twice). For each sample the three extracts were pooled and stored at −20°C until use. Before LC‐MS, the extracts were concentrated in a vacuum centrifuge at 50°C, and volumes were adjusted to 50 μl by adding 0.05% formic acid, filtered through a 0.45 um spin filter, and transferred to an LC autosampler vial. STATISTICAL ANALYSIS AND BIOLOGICAL PATHWAY ANALYSIS Cluster analysis of phosphopeptides and phosphosites was performed using hierarchical clustering. Phosphopeptide intensities were normalized to zero mean and unit variance for each phosphopeptide. Normalization of phosphopeptide intensities and cluster analyses were performed in R version 3.5.1. For comparative analyses, only high confidence class 1 phosphosites were considered. Aiming to distinguish a phosphosite and protein signature predictive of treatment outcome of sunitinib, differential expression patterns were analyzed using the Linear Models for Microarray and RNA-Seq Data (limma) package version 3.36.5 for R37,38 (filters: p < 0.05, fold change (FC) > 2, ≥ 30% data presence, i.e. there must be a non-zero value in at least 30% of samples in the group with highest abundance). Differential expression of proteins was analyzed using the filters: p < 0.05, FC > 2 and ≥ 50% data presence; here, with
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