Abstract
Approximately two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments. The absence of brain injury diagnostic endpoints was identified as a significant roadblock to TBI therapeutic development. To this end, our laboratory has studied mechanisms of cellular injury for biomarker discovery and possible therapeutic strategies. In this study, pooled naïve and injured cortical samples (48 h postinjury; rat controlled cortical impact model) were processed and analyzed using a differential neuroproteomics platform. Protein separation was performed using combined cation/anion exchange chromatography-PAGE. Differential proteins were then trypsinized and analyzed with reversed-phase LC-MSMS for protein identification and quantitative confirmation. The results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and alphaII-spectrin were found to be elevated after TBI. Differential changes in the above mentioned proteins were confirmed by quantitative immunoblotting. Results from this work provide insight into mechanisms of traumatic brain injury and yield putative biochemical markers to potentially facilitate patient management by monitoring the severity, progression, and treatment of injury.
Highlights
Two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments
The primary mechanical injury produces a robust pattern of necrotic cell death in close proximity to the impact site that is mediated by calpains, tein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CRMP-2, collapsin response mediator protein 2; MAP, microtubuleassociated protein; breakdown products (BDPs), breakdown product; 1D, one-dimensional; 2D, two-dimensional
Our hypothesis is that the cation/anion exchange (CAX) chromatography-PAGE/reversed-phase (RP) LC-MSMS (CAX-PAGE/RPLC-MSMS) platform will improve discovery of differential protein changes post-TBI and facilitate discovery of biochemical markers and possible therapeutic interventions
Summary
The biochemical mechanism that produces post-TBI changes in these proteins are not understood, leaving them as potential surrogate markers rather than true biochemical markers of known injury pathways To this end, breakdown products of proteolyzed proteins are of particular interest in neurotrauma as they provide a direct assessment of a known neurodegenerative mechanisms with the potential for therapeutic intervention. The primary mechanical injury produces a robust pattern of necrotic cell death in close proximity to the impact site that is mediated by calpains, tein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CRMP-2, collapsin response mediator protein 2; MAP, microtubuleassociated protein; BDP, breakdown product; 1D, one-dimensional; 2D, two-dimensional.
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