ITRAQ-Based Proteomics Reveals Plasma Biomarkers For Patients With Prolonged Disorder Of Consciousness

Background:Patients who survive severe traumatic brain injury(sTBI)may be left unconscious for at least a month,which is also called prolonged disorders of consciousness,including coma,vegetative state(VS)or minimally conscious state(MCS).Consciousness can be divided into wakefulness(i.e.,spontaneous or stimulated eye-opening cycle)and awareness(i.e.,perceptions of self or environment).Coma is a condition without signs of awareness nor wakefulness,rarely persist for longer than a month.Vegetative state is a condition with preserved eye-opening cycles but complete lack of awareness,while minimally conscious state is a condition showing inconsistent but reproducible awareness.Behavioral assessments have currently used as diagnostic tools,however,potentially leading to a high rate of misdiagnosis.How to develop effective and reliable methods to detect the loss of consciousness remains a major problem.Outcome of patients with disorders of consciousness was evaluated according to the extent of recovery of awareness.Previous studies have reported that the probability of recovery is less than 1%when it lasts longer than 3 months for non-traumatic vegetative state or 12 months for a traumatic vegetative state,which was also termed as"permanent vegetative state" or "chronic vegetative state".A series of questions concerning whether the patients with vegetative state should be treated or not have been raised for many years,which means low quality,meaningless of life but high financial burden to the family and society.However,it is still difficult for withdrawing treatment in patients with vegetative state.It is therefore necessary to define the patients with potential to recovery and take positive actions before the "permanent vegetative state".Striking new advances have been made in the diagnosis,prognosis,intervention and rehabilitation of prolonged disorder of consciousness within the few past decades.The neuroimaging and neuroelectrophysiological techniques have opened new avenues towards exploring the residual brain function.However,little was known about protein biomarkers and the molecular basis involved in prolonged disorder of consciousness was not clear.Proteomics techniques are cvurrently being used to detect differentially expressed proteins(DEPs)associated with pathological states of sTBI.The identification and quantification of novel potential biomarkers provide insight into diagnosis,monitoring,prognosis,and planning of treatment strategy in patients with prolonged disorder of consciousness.A combined isobaric tag for relative and absolute quantitation(iTRAQ)-liquid chromatography-tandem mass spectrometry(LC_MS/MS)-based proteomics approach,a powerful and sensitive proteomics tool,is used to scan a comprehensive differential proteome associated with prolonged pathological states of disease.Specific protein biomarkers for patients with prolonged disorder of consciousness not only helps to understand the evolution of disorder of consciousness caused by sTBI,but ean also be useful complements to clinical assessments.Aim:To examine the plasma protein markers in patients with prolonged disorder of consciousness caused by sTBI,we used the iTRAQ technique to perform the following investigations:(1)To identify differences in plasma protein expression between patients with prolonged disorder of consciousness and healthy individuals,to select potential protein biomarkers and to further verify and describe their clinical benefit.(2)To evaluate the effects of peripheral inflammatory ceytokines in patients with prolonged disorder of consciousness.Part Ⅰ:iTRAQ-based proteomic identification and functional characterization of differentially expressed plasma proteins in patients with prolonged disorder of consciousnessMethod:For the screening test,plasma samples were obtained at 3 time points(1,3 and 6 months)from 18 patients with prolonged disorder of consciousness after sTBI,and 6 healthy volunteers.A coupled isobaric tag for relative and absolute quantitation(iTRAQ)-based proteomics approach was used to screen differentially expressed proteins(DEPs)between patients and controls.An overall distribution pattern and fimctional enrichment analysis of these DEPs was performed by DAVID database.A protein-protein interaction(PPI)networks for DEPs between patients and controls were constructed,and the hub proteins were identified with an interaction degree>15.Candidate protein biomarkers were then tested in enzyme-linked immunosorbent assay(ELISA).Results:A total of 300 proteins containing at least one unique peptide were identified(False discovery rates[FDR]<1%).77,72 and 93 statistically significant DEPs were obtained from patients at 1 month,3 month,and 6 months,respectively.Of these altered proteins,32 proteins were consistently differentially expressed among different groups.Biological pathway analysis revealed that the DEPs were predominantly involved in complement cascade.C3,C9,and MBL2,three proteins from the complement cascade,were validated using ELISA.The result showed that proteins of complement cascade had significantly higher expression in patients with prolonged disorders of consciousness at 1,3,and 6 months compared with healthy controls.Conclusion:In this study,we used iTRAQ-based proteomics approach to identify DEPs in patients with prolonged disorders of consciousness and healthy individuals.Candidate proteins were then validated by ELISA,which may serve as potential biomarkers for further research.This study has shown higher expression of proteins of complement cascade,suggesting a persistent activation of complement cascade during the chronic unconscious stage after sTBI.Part Ⅱ:Peripheral inflammatory markers in patients with prolonged disorder of consciousness after severe traumatic brain injuryMethod:Patients with prolonged disorder of consciousness after sTBI were recruited at the rehabilitation units of Hangzhou Wujing Hospital during January 2015 to January 2018.A total of 101 cases and 22 healthy controls were included in this study,demographic characteristics and 5 ml peripheral blood were synchronously collected.The Coma Recovery Scale-Revised(CRS-R)score was used to quantify the severity,and recovery at 12 months was assessed by Glasgow Outcome Scale(GOS).The serum concentrations of IL-1β,IL-4,IL-6,IL-10,IL-13,and TNF-α were assessed using the Milliplex MAP Kit.Patients were randomly divided into a training group and a validation group.Then,conventional logistic regression and XGboost algorithm were used to construct a predictive model about the recovery of patients based on the peripheral inflammatory markers and related variables.Results:101 patients with prolonged disorder of consciousness and 22 healthy controls were enrolled in the study.At baseline,IL-6,IL-10,IL-13 and TNF-α showed significantly higher in patients with DOC compared with controls,while no difference in cytokine levels was shown between patients with vegetative state(VS)and patients with minimally conscious state(MCS).IL-10 and IL-13 were found corrected with CRS-R scores in patients with DOC.The predictive model was constructed through conventional logistic regression and XGboost-based machine learning.ROC analyses demonstrated a high accuracy to predict poor outcome with AUC of 0.883 for XGboost-based machine learning and 0.588 for conventional logistic regression model.Conclusion:In summary,this study showed a peripheral inflammatory response during the late unconscious period after severe traumatic brain injury.Although chronic inflammatory activity has not been well characterized,several peripheral inflammatory markers might offer a feasible way to investigate long-term pathophysiologic processes involved in prolonged disorder of consciousness after sTBI.Future larger studies are required to further elucidate the value of these inflammatory markers.