Brain Proteomic Study In Different Developmental Stages And The Study On Expression Level Of Neuroglobin In Serum

Objective: This study is a part of the Human Proteome Organization Brain Proteome Project (HUPO BPP) pilot study, which aims to obtaining a reliable database of mouse brain proteome, evaluating brain proteomes of three different age stages, and preparing to perform proteome studies of neural diseases for next step. Methods: The mouse brain samples were provided by HUPO BPP Committee, including C57/B16 female mouse brains of three different developmental stages of embryonic day 16, postnatal day 7 and of 8 weeks. The whole brain was used for tissue sample preparation following routine method. After then, two-dimensional electrophoresis was performed following with in-gel digestion. The selected protein spots were then identified by MALDI-TOF/TOF tandem mass spectrometry coupled with using Mascot Search engines to search the UniProt Database. Results: The 2D gel maps of mouse brains in all of the developmental stages were obtained. For quality control of the proteomic strategy used for the HUPO BPP pilot study, four protein, alpha enolase, phosphoprotein p19, and two actins, which expressed at same level in each developmental stage were successfully identified. Furthermore, to evaluating the performance of the proteomic approach used in this study, four down-regulated proteins, C14orf166 homolog, 28× 10~3 heat- and acid-stable phosphoprotein, 40S ribosomal protein S3a and 3-mercaptopyruvate sulfurtransferase were successfully identified. Conclusions: The development-related proteins such as alpha enolase and protein C14orf166 homolog, are also supported by literature revealing the relative to ontogenesis or brain development. These differentially expressed proteins are important for discovering the molecular mechanisms of brain development, and reference to future proteome studies of neural diseases. According to which, the proteomic approaches have been established in our lab and it is now ready for performing the next step of the HUPO BPP studies.Neuroglobin (NGB) is a newly discovered protein localized in neurons of the central and peripheral nervous systems in vertebrates. It functions to bind, store, and facilitate the utilization of oxygen in neuronal cells, which improves the tolerance ability of brain to hypoxia. To establish the quantitative method to measure NGB in serum by Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), serums were measured from normal newborn, fetal distress, asphyxia and premature infants, and observed the change trend of NGB to evaluate serum NGB as a sensitivity and validity potential biomarker for brain injury.Results: l.The linearity correlation modulus of ELISA standard curvilinear was 0.9994983, and the lowest detected level was 28.7ng/ml, both individual variance and batch variance quotient were less 15%. 2. The average NGB level in umbilical cord artery serum of normal newborn was 27.35±4.41ng/ml, and no difference in gender (P>0.05). 3. The NGB in umbilical cord artery serum of fetal distress presented higher levels compared with normal newborn (P<0.01);the NGB in umbilical cord artery serum of both asphyxia and premature infants also presented higher levels compared with normal newborn (P<0.05, P<0.01). 4. Using lung surfactant to premature infants for avoiding neonatal respiratory distress syndrome, the treatment group had a lower NGB level in relation to the control (the group of no treatment premature infants) after born 6 hours (P<0.05), and there were no difference in two groups after born 1 hour and 3 hours (P>0.05).Conclusions: 1. Using double-antibody sandwich ELISA to detect NGB in serum, which is credibility, sensitivity and repeatability, large quantities of samples would be measured by this standard approache. 2. The NGB in umbilicalcord artery serum of normal newborn had a low level even undetected, and no difference in gender. 3. Both fetal distress as long term hypoxia and birth asphyxia as acute hypoxia could increase the levels of NGB in umbilical cord artery serum, the result which was the same as that of animal experiments indicted probability of hypoxic brain damage. And the increased NGB levels of premature infants maybe suggested the incidence of cerebral palsy as one of the danger factors. 4. Using lung surfactant to premature infants early, the treatment group had a lower NGB level compared with the control after born 6 hours, it was shown that early treatment had an important clinical significance to reduce premature infants hypoxic brain damage.