Table 3.

Pre-clinical utilization of neuroproteomics approaches in traumatic brain injury studies.

Animal subject	Injury type	Media tested	Techniques	Results	Validation	Reference
Sprague–Dawley rats	Fluid-percussion brain injury	CSF	2D-PAGE, HPLC, and MALDI-TOF/MS	60 proteins and their proteolytic fragments were identified to be released from cortical neurons associated with neurodegeneration. 14–3-3ξ, 14–3-3β, and 17-kDa fragments of tau were described as possible markers for acute brain injury.	Western blot analysis of conditioned media confirmed that 14–3-3ξ, 14–3-3β, CRMP-2, CRMP-4, and GAP43 were released in response to necrotic neurodegeneration.	(206)
Sprague–Dawley rats (n = 7)	Controlled cortical impact (CCI)	Brain tissue	Combined cation/anion exchange chromatography-PAGE, and reversed-phase LC–MS/MS	59 differential protein components were identified, 21 decreased, and 38 increased significantly in TBI. The former included collapsing response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely, the latter included C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and αII-spectrin.	Western blot analysis validated changes in protein levels and breakdown products.	(20)
Sprague–Dawley rats	CCI	Brain Tissue	Gel electrophoresis and MALDI-TOF/MS	Several mitochondrial proteins have undergone oxidative modification in the cortex and the hippocampus. In the cortex, the proteins are pyruvate dehydrogenase, voltage-dependent anion channel-2 (VDAC-2), fumarate hydratase 1, ATP synthase H+ transporting F1 alpha subunit. As for the hippocampus, the proteins are cytochrome C oxidase Va, isovaleryl coenzyme A dehydrogenase, enolase-1, and glyceraldehyde-3-phosphate dehydrogenase.	Western blotting validation was conducted for VDAC-2.	(266)
Sprague–Dawley rats (control/CCI male rats n = 7 and 9, respectively; control/CCI female rats n = 9 and 7, respectively)	CCI	Brain tissue	Fluid-phase isoelectric focusing, MALDI-TOF/MS, and immunohistochemistry	After injury, the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein carbonylation. The most susceptible for postinjury carbonylation were astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence. Upon proteomics analysis, glial fibrillary acidic protein, dihydropyrimidinase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A were the most affected by carbonylation in response to TBI.	N/A	(210)
Sprague Dawley rats (n = 73)	CCI After injury, the rats inhaled normoxic (22% O2) or Hypoxic (11% O2) air.	Serum	Targeted antibody suspension bead array	During the first days of injury, complement factor 9 (C9), complement factor B (CFB), and aldolase c (ALDOC) were detected at higher levels (value of p <0.01). In weeks period, hypoxia-inducing factor (HIF)1α, amyloid precursor protein (APP), and WBSCR17 were increased (value of p <0.05).	N/A	(209)
Sprague Dawley rats	Free-falling hammer (450 g) onto a stainless-steel disk attached to the rat’s head was used to induce injury	Cerebral brain tissue	iTRAQ and LC–MS/MS	1858 proteins were identified and quantified, where the comparative analysis identified 10 candidate proteins worth exploring. Out of these, citrate synthase, which binds to the inner membrane surface of the mitochondria and plays a critical role in the central metabolic pathway of aerobic organisms, was significantly downregulated 1 h post-injury. Similarly, synaptosomal-associated protein 25(Snap25) was downregulated 3d after the injury. Snap25 not only plays an important role in the regulation of synaptic vesicle exocytosis but it is also involved in axonal elongation.	Citrate synthase (CS), synaptosomal-associated protein 25 (Snap25), microtubule-associated protein 1B (MAP1B), and Rho-associated protein kinase 2 (Rock2), were validated by Western blot and immunohistochemistry analyzes.	(267)
Adult male Yucatan miniature and Yorkshire swines (n = 4 per group)	Blast tube was used to simulate free-field blast at a single moderate overpressure exposure of 40–52 psi	Frontal cortex brain tissue	LP-IEF, SDS-PAGE, and LC–MS/MS	Blast exposure was shown to affect deamination patterns in the brain proteome. 6 different proteins were shown to have undergone deamination, including GABA transaminase, aconitate hydratase, GFAP, Glutathione S-transferase, Histone H4, and vimentin. Additionally, the results showed a significant elevation of IgGs in the cerebral cortex.	Protein panels and IgG levels were validated by Western Blotting.	(268)
Male C57BL/6 J mice	Open-field low-intensity blast injury at a magnitude of 82 kPa	Brain tissue	TMT-tagging, LC–MS/MS	Mouse subjected to the blast displayed increased levels of tau protein phosphorylation 3 h to 24 h after injury. Analysis of phosphoproteins after the blast showed downregulation of 29 proteins and upregulation of only one protein at 3 h. However, four were downregulated, and 17 were upregulated at 24 h. The phosphoproteins with the largest increase include Add1, Camk2b, Syt1, and Stmn1. Ap3b2, Sgip1, Basp1, and Rph3a were among those significantly downregulated phosphoproteins.	N/A	(269)
Male Sprague Dawley rats	custom-designed device of metallic pendulum-striker was used to induce mTBI	Prefrontal cortex tissue	iTRAQ, HRPH, and LC–MS/MS	Analysis showed that 237 proteins were significantly changed in mTBI groups compared to the sham injury group. Significantly, two proteins, Pde10a and Gnal, which are involved in cAMP signal pathway, were suggested to play a role in mTBI pathogenesis. Both proteins were acutely upregulated after the injury and did not return to baseline levels until 6 months after the injury.	Western analysis of Pde10a and GnaI was done for validation.	(150)
Long-Evans rats	Repeated mild lateral fluid percussion	Plasma	Reverse Phase Protein Microarray	Plasma biomarkers indicated axonal damage, astroglia damage, oxidative stress, and metabolic dysfunction. There was a significant increase in GFAP (p = 0.029), 4-HNE (p = 0.003), NF-H (p = 0.004), and ceruloplasmin (p = 0.006) after injury. However, VEGF levels were found to be decreased (p = 0.002).	N/A	(270)
Male C57BL/6 J mice	Open-field low-intensity blast injury at a magnitude of 46.6 kPa	Brain tissue	TMT, LC–MS/MS	Results showed changes in 2216 and 459 phosphorylated proteins at various time points after the blast. Important pathways involved included evidence of mitochondrial dysfunction, oxidative stress, axonal/cytoskeletal/synaptic dysregulation, and neurodegeneration. Bioinformatic analysis identified blast-induced events related to cellular growth/development/movement/assembly and cell-to-cell signaling interactions. Notably, mitochondrial dysfunctions included impaired fission-fusion dynamics, diminished mitophagy, decreased oxidative phosphorylation, and compensated respiration-relevant enzyme activities.	Changes in mitochondrial markers associated with oxidative stress and fission-fusion dynamics at different time points post-injury were validated through western blotting.	(271)
Male Sprague–Dawley rats	Mild lateral fluid percussion injury (FPI), traumatic axonal injury (TAI)	Brain tissue	DIA LC–MS/MS	Results showed that developmental changes and TBI can cause modifications in axonal microtubules (MTs) through post-translational changes in tubulin and MT-associated proteins (MAPs) such as tau and MAP6. Degenerating axons show instability and depolymerization, while nearby axons without degenerating morphologies show enhanced MT stabilization. Further study is needed in this area.	Changes in tau and MAP6 expressions during development and after TBI, and also axon degeneration changes and tubulin PTMs were validated by western blotting, and IHC staining.	(163)
Male Long-Evans rats	Awake closed head injury (ACHI), repeated mild TBI	Tissue and Serum	DIA mass spectrometry, MRI	Repeated mTBI rats had acute cognitive deficits and prolonged sensorimotor impairments. Serum NfL was elevated at 7 days post-injury, correlating with sensorimotor deficits. Several hippocampal proteins were altered by repeated mTBI, including those associated with energy metabolism, neuroinflammation, and impaired neurogenic capacity. Diffusion MRI analysis at 3.5 months found widespread reductions in white matter integrity.	N/A	(161)
Adult male Sprague–Dawley rats	Mild–moderate controlled cortical impact (CCI)	Brain tissue	Ion mobility DIA mass spectrometry, Immunoblot analysis, Immunofluorescence microscopy	This study used artificial neural network and functional enrichment analyzes to identify ion transporters that could help address abnormal GABAergic transmission and delayed decline following brain injury. They focused on KCC2 or SLC12A5, and tested a KCC2-selective modulator CLP290. The modulator was effective at restoring lost KCC2 localization and improving somato-sensory behavioral tasks, but timing of administration was crucial. Results suggest the importance of post-translational characterization in developing TBI treatments, and the promise of KCC2-targeted CLP290 intervention for positive functional recovery after brain injury.	Validation of the escalated loss of KCC2 through the first week was confirmed through immunoblotting. However, the decrease in total protein was observed to be delayed until the second day, which was affirmed across two independent antibodies.	(164)
Adult C57BL/6 mice	Stab wound injury affecting gray matter (GM) and white matter (WM)	Brain tissue cerebral cortex	Confocal laser scanning, Isolation and fluorescence-activated cell sorting, DIA mass spectrometry	The study examined glial reactivity in regions affected by WM and GM injuries and compared the impact of WM injury on reactive gliosis in the GM. Results showed that microglia proliferation increased in the WM compared to GM in the GM+ injury, while proliferating astrocytes were more abundant in the GM. WM lesion strongly influenced the proliferation of GM glial cells, particularly at early stages post-lesion. The study also found that NG2 glia proliferation was decreased in the GM+ compared to the GM-lesion condition and that MIF regulates NG2 glia proliferation.	N/A	(162)