Ultra-deep Brain Tyrosine Phosphoproteomic Profiling by SH2-superbinder

Addiction is a complex disease that involves drug-induced changes in synaptic plasticity due to alterations in gene transcription, protein synthesis, and cell signaling. Evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include phosphorylation mediated signaling by a subset of specific protein kinases. Addiction to drugs such as nicotine and cocaine primarily affects brain and the limbic system (ventral tegmental area (VTA), striatum, and NAc) that have rich protein kinase expression. Research has established that several kinases such as cAMP-dependent protein kinase, cyclin-dependent protein kinase, protein kinase C, calcium/calmodulin-dependent protein kinase II, Src, and Fyn tyrosine kinase play a role in the neuro-pathology of drug addiction. Identifying phospho-mediated signaling pathways that contribute to the addicted state may provide novel approaches for treatment of drug addiction. The analysis of protein phosphorylation sites has largely been restricted to studies at the single-protein level. Recently, larger-scale MS-based analyses have emerged. However, such studies have been challenging due to the immaturity of methods to enrich for low-abundance phosphoproteins or phosphopeptides. In this pilot project we will develop a high throughput, novel, convenient, sensitive, and cost-effective affinity reagent (SH2 superbinder) that will have wide applications in protein tyrosine phosphorylation analyses in drug addiction and neuronal signaling. This pilot project has two major aims: 1): Evaluate the specificity of SH2 superbinder in pTyr peptide enrichment in the rat brain. 2): Next, we will discover how cocaine exposure changes the tissue specific pTyr-proteome by comparing cocaine treated vs control rats. Considering that pTyr analysis in these small brain regions (Striatum, VTA, NAc) is difficult, we will address this technical challenge using a cutting-edge discovery proteomics technology that uses tandem mass tag (TMT) labeling.