| Objectives:A high mortality rate about 30%-50%is associated with severe traumatic brain injury (TBI), few survived but also disabled. TBI is a major health problem and produces a considerable medical expense to the society and the family worldwide. Despite many researches focus on the pathophysiology and neuroprotective mechanism, there are currently no truly effective therapies for TBI.During our research on "Proteomic analysis of the differential protein expression of the human cerebral cortex after acute severe traumatic brain injury" supported by National Natural Science Foundation of China (No.30271331),138 protein spots were found with altered expression,64 distinct proteins were identified by mass spectroscopy. In this study. Cerebrospinal fluid (CSF) from patients with traumatic brain injury was analyzed by differential proteome technique, aiming to identify the brain-derived differential proteins.Methods:In first section, CSF was gethered from a pool of 10 patients with TBI underwent ICP monitor by extraventricular drainage. CSF sample was desalted and concentrated by ice-cold acetone, thus meeting the requirements of having a concentration>50mg/mL necessary for using the ProteoMinerTM large-capacity kit. Protein equalizer technology (ProteoMinerTM) has been used for the enrichment of low abundant protein biomarkers for TBI and simultaneous reducement of the high abundant proteins in human CSF. The bound proteins were separated by two-dimensional electrophoresis. The silver-stained 2-D gels were scanned, and then analyzed by PDQuest 7.0 software package. We got these high-producible 2D gel proteome maps of human CSF with reducement of high abundant proteins.In second section, large volumes of CSF were obtained within 24 hours from a pool of 30 patients with TBI underwent intracranial pressure (ICP) monitor via extraventricular drainage (EVD). Another pool of CSF samples from 8 patients was used as the control. which sufferring from subarachnoid hemorrhage without hemotoma in the parenchyma caused by a ruptured aneurysm. and an EVD was performed to withdraw CSF. Each sample obtained and centrifuged at 2000×g for 10 min to eliminate cells and other insoluble materials. The samples were then stored at-80?. Low abundant proteins were enriched as mentioned in the first section.50?g bound proteins from each group and the internal standard were cross-labled with different CyDyes. Two-dimensional differential in-gel electrophoresis (2D-DIGE) was performed to get the differential expression protein spots with DeCyder v.5.02 software. These protein spots were digested with trypsin and Peptide Mass Fingerprint (PMF) was analyzed using a 4800 matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI TOF/TOFTM) Analyzer (Applied Biosystems). Protein identification by PMF was performed using the GPS ExplorerTM V3.5 software by searching in the International Protein Index (IPI) human database V3.23. And data mining about the function of these proteins were also conducted.Results:In first section, after the treatment by ProteoMinerTM column, the total number of spots in the 2-D gel was 1432±85, whereas 1123±114 spots were observed on the 2-D gels treated by ice-cold acetone. There were significant differences between the two prefractionation methods (P<0.05, F=8.885). Using ProteoMinerTM clearly improved the resolution and increased the intensity of the low abundance proteins compared with 2-D analysis of acetone precipitated CSF samples, and simultaneously reduced the high abundant proteins. Thus this prefractionation method was applicable for further research.In second section, low abundant proteins in the CSF of the TBI group and the control group were enriched by ProteoMiner, and separated by 2D-DIGE, the map of CSF proteins was clear. The results of biological variation analysis (BVA) on the TBI group and the control group showed 169 protein spots with an expression change over 1.5-fold.98 up-regulated and 71 down-regulated protein spots were selected for further analysis with MALDI-TOF mass spectrometry.47 spots corresponded to 36 distinct proteins were identified. These identified proteins have been grouped into different categories based on their possible biofunctions:cytoskeleton. intermediary metabolism, signaling transduction, stress response, protein metabolism and unknown. Among these proteins, the ceruloplasmin, parvalbumin, Krueppel-like factor 6. phospholipid transfer protein, SLC25A23, Bromodomain containing protein 1 and F-box protein 7 are associated with central nervous system (CNS) diseases. This study first show expression changes in these proteins in acute TBI patients.Conclusion:The mechanical injury to the brain apparently affects the protein profile of CSF in the acute phase, and makes CSF a better window to study TBI and other CNS diseases. The use of the protein equalizer technology (ProteoMinerTM) increased 2-DE gel resolution and enabled detection of low abundance proteins compared with the precipitation technique. Up-regulation and down-regulation of proteins existed at the same time. Although further confirmation may need, these differential expressed proteins may become the biomarker for evaluating injury severity or predicting the clinical outcome. |
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