Protocol for integrated phosphoproteomic and proteomic analysis of the mouse ventral tegmental area with functional and kinase motif enrichment.

Abstract

We present a protocol for label-free proteomic and phosphoproteomic analysis of the mouse ventral tegmental area (VTA). We describe steps for combining region-specific biopsy-punch collection, parallel proteomic and phosphoproteomic processing, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and downstream enrichment analysis. We detail procedures for SynGO-based synaptic localization, g:Profiler/DAVID pathway enrichment, and iceLogo/pLogo motif analysis. This protocol enables quantitative assessment of protein abundance, phosphorylation dynamics, and functional interpretation from limited brain region samples. For complete details on the use and execution of this protocol, please refer to Diezel et al.1.