Cells possess enzymes and proteins that can write (kinase), erase (phosphatase), and read (phospho-specific binding domains) phosphorylation modifications across the proteome to form a complex signaling system for regulating proliferation, differentiation, and transcriptional activation. Aberrant phosphotyrosine (pTyr) signaling underlie many human diseases and the identification of molecular components of pTyr processing has uncovered fundamental principles of signal transduction and furnished new targets for therapy. A key example is the oncogenic BCR-ABL fusion protein that exhibits constitutive tyrosine kinase activity resulting in excessive pTyr modifications to transform cells in cancer. The development of small molecule (GleevecTM) and antibody (HerceptinTM) therapeutics that target pTyr signaling continues to transform anti-cancer treatment options in the clinic. Despite enormous clinical potential, a critical barrier that remains in the biomedical field is the ability to assign site specific phosphorylation events to functional changes for therapeutic targeting in human disease. Technologies capable of overcoming the low abundance and substoichiometric phosphorylation-site occupancy of phosphoproteins are needed to address the challenge of functional phosphoproteomic profiling. This is especially true for phosphorylation of Tyr. Compared with phospho-Ser and -Thr, pTyr modifications represent a rare subset (~1%) of the human phosphoproteome. The objective of the proposed studies is to apply a site trapping by covalent probes (dubbed SiteTraP) methodology to assign substrate specificity to the PTP oncogene, tyrosine-protein phosphatase non-receptor type 11 (SHP2). The significance of the proposed studies is development of a chemical proteomics strategy to assign substrate specificity – at the protein and pTyr site level – to individual PTPs directly on native proteins in lysate and cellular studies. We will test 2 independent, yet related specific aims directed at benchmarking: (Aim 1) pTyr specificity of SiteTraP in the presence of phospho-Ser and -Thr in complex proteomes, (Aim 1) Sensitivity of SiteTraP for capturing SHP2-specific dephosphorylation of pTyr sites in complex proteomes, (Aim 2) Capability of SiteTraP for capturing global pTyr activation in PTP-disrupted live cells, and (Aim 2) Sensitivity and specificity of SiteTraP for assigning substrate specificity to SHP2 and determining how SHP2 inhibitors disrupt these networks in live cells. Broadly, our proposed studies will be important for the biomedical community by 1) guiding PTP inhibitor development for targeting specific pTyr modifications in human disease, 2) revealing differences in active site and allosteric SHP2 inhibitor mode of action for basic and translational understanding of PTP pharmacology, and 3) gain a deeper understanding of PTP-substrate networks and regulation in live cells.