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Protein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions,subcellular localization,and ultimately cellular functions.Here we introduce several proteomic strategies based on chemical derivatization to study signaling network.In the first step,we have utilized a novel soluble nanopolymer-based approach,termed PolyMAC (Polymer-based Metal ion Affinity Capture),for homegeneousphosphopeptide isolation with high efficiency and specificity.Furthermore,we have devised a proteomic strategy to identify direct substrates of protein kinases through Kinase Assay LlnkedPhosphoproteomics (KALIP),which combines a sensitive stable isotope labeled kinase reaction with quantitative phosphoproteomics.Finally a hybrid platform is being developed through array-based phosphorylation visualization followed by mass spectrometry for differential phosphorylation analysis in order to focus on relevant phosphorylation events without prior large-scale mass spectrometry-based phosphoproteome profiling.