Systems-wide Analysis of a Phosphatase Knock-down by Quantitative Proteomics and

Signal transduction in metazoans regulates almost all as-pects of biological function, and aberrant signaling is in-volved in many diseases. Perturbations in phosphoryla-tion-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific an-tibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phospha-tase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data