Deep Proteome Profiling of Rat Dorsal Striatal Synaptoneurosomes Following Methamphetamine Exposure

Understanding how drug exposure alters synaptic proteins is essential for uncovering the molecular mechanisms underlying addiction and identifying new therapeutic targets. Synaptoneurosomes (SNs), subcellular fractions enriched in pre- and postsynaptic components, provide a powerful resource for studying synaptic adaptations in the brain. Here, we applied a deep, discovery-based proteomic strategy to profile SNs isolated from the dorsal striatum (DS) of rats 1 h after acute methamphetamine (Meth) administration. To maximize proteome coverage, we combined tandem mass tag (TMT) labeling, offline high-pH fractionation, and low-pH nanoflow liquid chromatography coupled to high-resolution (orbitrap) mass spectrometry (HRMS). This approach enabled identification of ∼6100 cytosolic and membrane proteins from ∼500 ng of SN proteome digest─representing the most comprehensive DS SN proteome reported to date. A comparative analysis between Meth-treated and saline control animals revealed 147 differentially abundant proteins (81 with increased abundance; 66 with decreased abundance) enriched in pathways related to dopamine biosynthesis, synaptic vesicle cycling, mitochondrial energy metabolism, and proteasomal degradation. These findings highlight coordinated molecular remodeling of striatal synapses in response to Meth and demonstrate the utility of deep SN proteomics in addiction research. Deep, low-input SN proteomics nominates vesicle acidification, mitochondrial ATP supply, and proteasome function as testable pathway-level targets in addiction.