Quantitative Proteomics Analysis To Identify Differentially Expressed Proteins In Rats With Diffuse Axonal Injury Using ITRAQ Coupled LC-MS/MS

Objective This study employed an established diffuse axonal injury(DAI)animal model in conjunction with an isobaric tag for relative and absolute quantification(iTRAQ)-based protein identification/quantification approach to elucidate the detailed mechanisms of axonal injury,and to identify high sensitive and specific biomarkers of DAI.These findings will further an understanding of the pathophysiological mechanisms underlying the substantial mortality and specific symptoms in patients with DAI.Furthermore,the identified differentially expressed proteins may serve as reliable DAI biomarkers and aid in early diagnosis and treatment.Methods DAI was induced in animals using an injury model adapted from Marmarou A et al.DAI animal models were first histopathological validated using HE staining and Bielschowsky silver staining.Animals were randomized into 8 groups(N=9 rats/group): control group [with two subgroups: sham group and sham-injured group];injury group [with six subgroups: rats that immediately died after injury and those sacrificed at 1h,6 h,1 d,3 d and 7 d post-injury].An iTRAQ-based proteomic approachwas utilized to identify several differentially expressed proteins in DAI cerebral tissues and plasma tissues.Bioinformatics analysis was further applied and the differentially expressed proteins proteins were successfully confirmed by Western blot and/or immunohistochemistry analysis.Results A total of 1858 proteins were identified and quantified in DAI cerebral tissues.Comparative analysis identified ten candidate proteins that warranted further examination.Of the ten candidate DAI biomarkers,four proteins,citrate synthase(CS),synaptosomal-associated protein 25(Snap25),microtubule-associated protein 1B(MAP1B)and Rho-associated protein kinase 2(Rock2),were validated by subsequent Western blot and/or immunohistochemistry analyses.A total of 374 proteins were identified and quantified in DAI plasma samples.Of these,44,37,33,36 and 36 proteins were identified as differentially expressed in rat plasma samples 1 h,6 h,1d,3 d and 7 d after DAI relative to the controls,respectively.Two candidate DAI biomarkers,Glyceraldehyde-3-phosphate dehydrogenase(Gapdh)and Hemopexin(Hpx),were validated by subsequent Western blot analyses.Conclusions Our studies identified several differentially expressed proteins that not only offers us new insights into the pathophysiological mechanisms of axonal injury in DAI,but also may be a powerful tool for early diagnosis.