Phosphoproteomic analysis reveals NPM-ALK–mediated changes in metabolic pathways. (A) Experimental strategy used for phosphoproteomic and metabolomic analysis of ALK. NPM-ALK⁺ cell lines were exposed to ALK inhibitor or DMSO. Phosphorylated peptides were enriched with metal oxide affinity chromatography (MOAC) followed by immunoprecipitation with pY antibodies and subjected to tandem MS analysis (biological triplicate). For the metabolomics, the cell lysates were subjected to LC-MS in both positive and negative mode, as well as gas chromatography MS (GC-MS). (B) The phosphoproteomic data plotted as number of proteins (frequency) vs the z-score based on change in spectral counts in response to ALK inhibition (CEP). A significance threshold was set at |z|≥ 1. (C) GO term enrichment analysis using Database for Annotation, Visualization and Integrated Discovery applied to the proteins that changed in spectral counts following ALK inhibiton. A subset of the significant terms is displayed and ranked by –log(p), the natural log of the P value. (D) List of GO terms that comprise the metabolic processes term.