Using an experimental TBI rat model of mild/moderate Controlled Cortical Impact (CCI) injury, we combined large-scale proteomics identification and relative quantification using Spatially-Resolved Microproteomics with MALDI MS Imaging of Lipids. Spatially by studying different regions in the brain post injury in a coronal view, with main focus on the injury site itself. Temporally by studying the acute and subacute phase post injury, including injured rat brains at 1 day, 3 days, 7 days, and 10 days post injury. Direct on-tissue micro-digestion followed by micoextraction from 1 mm2 surface area within the injured cortical tissue were subjected to LC-MS & MS/MS analysis using HR MS. In addition, several identified potential biomarkers within our study were used to stimulate dorsal root ganglion (DRG), astrocyte, and macrophage cell lines to obtain a better understanding of their role and contribution in the injury.