小鼠不同发育阶段脑蛋白质组学初步研究

Preliminary proteomic study on the mouse brain in different developmental stages

目的:本研究为国际脑蛋白质组计划(HUPO BPP)的预实验部分,旨在获得可靠的小鼠脑蛋白质组数据库,比较和评估小鼠不同发育阶段的脑蛋白质组,为下一步进行神经系统疾病的蛋白质组学研究奠定基础。方法:样品来源于国际脑蛋白质组委员会所提供的胚胎16 d、出生后7 d及生后60 d等3个发育时期的C57/B l6雌性小鼠脑组织。常规方法提取组织总蛋白,采用固相pH梯度的二维电泳分离和串联质谱分析等进行鉴定,使用M ascot软件搜索Un iProt数据库,鉴定蛋白种类。结果:获得3个不同发育时期小鼠脑组织蛋白质表达谱,完成国际脑蛋白质组计划的预实验所要求的质量控制,鉴定出各发育阶段均有稳定表达的4个蛋白点,包括α烯醇酶、磷蛋白质P19及2个肌动蛋白。经进一步评估后鉴定出随年龄增大而丰度减低的C14orf166同源蛋白、28×103热/酸稳定的磷蛋白、3-巯基丙酮酸硫基转移酶、40S核糖体蛋白S3 a等4个蛋白。其中α烯醇酶及C14orf166同源蛋白曾有文献报道与神经发育及组织发生密切相关。结论:蛋白质组学的方法为脑发育研究提供了有参考价值的数据,其差异蛋白的发现可促进对神经发育机制的了解,并将为后续启动神经系统疾病蛋白质组学研究提供参考。

Objective： This study is a part of the Human Proteome Organisation Brain Proteome Project（ HUPO BPP） in pilot phase, which aims at obtaining a reliable database of mouse brain proteomc ,evaluating brain proteomes at three different age stages, and preparing to perform proteome studies of neural diseases in next step. Methods： The mouse brain sampies were provided by HUPO BPP Committee, including C57/B16 female mouse brains of three different developmental stages i.e. embryonic day 16, postnatal day 7 and 8 weeks. The whole brain was used for tissue sample preparation using routine method. After that, two-dimensional electrophoresis was performed following in-gel digestion. The selected protein spots were then identified by MALDI-TOF/TOF tandem mass spectrometry coupled with Mascot Search engines to search the UniProt Database. Results： The 2D gel maps of mouse brains at all developmental stages were obtained. For quality control of the proteomic strategy used for the HUPO BPP pilot study, four proteins, i.e. alpha enolase, phosphoprotein P19, and two actins, which expressed at same level in each developmental stage were successfully identified. Furthermore, to evaluate the performance of the proteomic approach used in this study, four down-regulated proteins, i.e. C14orf166 homolog, 28 ×10^3 heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40S ribosomal protein S3a, were successfully identified. Conclusion： The development-related proteins such as alpha enolase and protein C14orf166 homolog, are supported by literature which revealed their relation to ontogenesis or brain development. These differentially expressed proteins are important for discovering the molecular mechanisms of brain development, and provide reference to fu-ture proteome studies of neural diseases. According to the results of present study, the proteomic approaches have been established in our lab and it is now ready for performing the next step of the HUPO BPP studies.