Proteomic Analysis Of The Differential Protein Expression Of The Human Cerebral Cortex After Acute Severe Traumatic Brain Injury

Objection: Traumatic brain injury (TBI) is a serious clinical problem associated with a high mortality rate and critical pathophysiological responses. The recent studies have suggested that primary TBI initiates a cascade of molecular and cellular events which include changes in gene and protein expression, resulting in neurons dysfunction and/or death.Proteomics is the science and methodology of the study of the proteome, which include all proteins expressed in a particular cell or tissue in a special time point. Proteomics technology mainly consists of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate the protein mixtures and mass spectrometry (MS) to analyze and identify the protein. Proteomics provide us a useful, large-scale, high-throughput tool to investigate the protein mixtures.Prior to research the differential proteomics of human brain, we must construct a two-dimensional electrophoresis technique system suitable for the human brain cortex in first section. We can get a high-reproducible 2D proteome map of human cortex based on this technique system.In second section, to better understand the mechanism underlying the acute severe traumatic brain injury, the differential proteomic analysis on human injured cortex at the different post-trauma time-point was performed using two dimensional gel electrophoresis followed by mass spectrometry.The difference between moderate TBI and severe TBI is so great that the clinical symptom, neurological deficits, and prognosis appear variable. So the aim of our third section is to analysis the differential protein expression changes between mild and severe TBI samples with proteomic technique including two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.Method: In first section we extracted the total proteins from all of the injured cortex tissues of human brain. We separated the total proteins with two dimensional electrophoresis including IEF and SDS-PAGE, stained the 2D gels with Coomassiae brilliant blue R-350 and nitrate silver. We got the high-producible 2D gel proteome maps of human brain cortex.In second section, we obtained the several brain injury brain samples, which were separated into experimental groups at the different post-trauma time-point. Two-dimensional gel electrophoresis was performed to get the differential expression protein spots with image analysis software, and these protein spots were digested with trypsin, were identified by MALDI-TOF MS. To data of the differential proteins, we may know the biological characters of proteins in the pathological conditions. We also may find a novel biomarker of traumatic brain injury, and the new therapy target.In third section, the aim of our study is to analysis the differential protein expression changes between moderate and severe TBI samples. For this purpose, we obtained the several brain injured cortex samples, which were separated into moderate and severe two groups by GCS. These differential protein spots following to digest with trypsin, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadropole time-of-flight mass spectrometry (ESI-Q-TOF2 MS).Result: In first section, we got the high-producible 2D gel proteome maps of human brain cortex, hi second section, we have found 138 differential protein spots significantly with the analysis of ImageMaster 5.0 software, then they were identified by MALDI-TOF MS. In total, 82 protein spots were identified, which were the products of 66 proteins. The identified proteins have been grouped into different categories based on their possible biofunctions: cytoskeleton, intermediary metabolism, electron transport, signaling transduction, nucleic acidsynthesis, stress response, protein synthesis and turnover, transporter and neurotransmission, other, and unknown. The most abundant proteins were intermediary metabolism (n=ll). During the acute early 8 hours phase of post trauma, only few proteins (10 protein spots) were over expression than the control sample. Most of the differential proteins appeared fluctuant change trends, the mean expression levels of them were lower than the control level. Through surveying the current brain proteome research reports, we found the four proteins which have not been reported in the human TBI research. They are proteins, septin 11, integrin-linked kinase, PEA15, synapsin II isoform Ila.In third section, we have found 12 significantly differenial protein spots (p<0.05), which were idnentified by MALDI-TOF MS or ESI-Q-TOF2 MS. In total, 12 protein spots were identified, which were the products of 10 proteins. 9 spots, 6 kinds of proteins (alpha-enolase, pyridoxine 5'-phosphate oxidase, triosephosphate isomerase, neuronal protein NP25, crystallin, stathmin-1) were over-expression in severe TBI group than moderate group. 3 spots, 4 proteins (HSPC108, glutathione S-transferase, 5'3'-nucleotidase, proapolipoprotein) were over-expression in moderate TBI group.Discussion: hi our study, we found the expression of the proteins appeared variable changes. In the time point 2h and 3h of post trauma, since the primary brain injury, almost 76% protein spots were lower than the control one. As the response of the body compensation, the expression of the protein synthesis begins to rise. 53 spots reached the abundance peak in the 4-6 hours post trauma, mainly include involved metabolism, protein synthesis and turnover, cytoskeleton proteins. But at the 8 hour, most of the abundance of protein spots declined again, the reason may be the body decompensation response. The body cannot repair the injured tissues and prevent from the exacerbating process.Many important proteins were represented with proteins showing a statistical significant change. It is likely that with a large sample size other identified...