20S蛋白酶体快速分离和异质性研究及蛋白质体外甲基化修饰和行为表征

【作者】 陈国强；

【导师】 李智立；

【作者基本信息】 中国协和医科大学 ， 生物物理学， 2010， 博士

【摘要】 20S蛋白酶体(core particle, CP)是一个相对分子量约为700 kDa的蛋白质复合体,由14种不同亚基组成,分别称为α1～α7,β1～β7,并共同形成一个桶状结构α7β7β7α7。在先前的研究中,CP的分离主要依赖抗体、液相色谱以及亲合标签纯化等技术。在本研究中,我们报道了一种简单快速、经济、有效的CP分离方法。该方法主要涉及多步超速离心和非变性聚丙烯酰胺凝胶电泳技术；然后,利用二维电泳结合质谱技术对分离得到的CP进行了鉴定和分析,并对不同组织细胞来源的CP进行了蛋白酶体异质性探讨。该方法不仅适合于大通量(来自红细胞)和小规模的CP(来自培养细胞)纯化,亦适合于分离不同组织细胞来源的CP。此外,我们在纯化过程中发展了一种新型的直接三维凝胶电泳技术(direct three-dimensional gel electrophoresis, d3-DE)。实验结果表明,该分离方法对CP以及其它蛋白复合体的分离具有较大的学术价值。蛋白质甲基化修饰对蛋白质行使特定功能具有重要作用。除常见的精氨酸和赖氨酸甲基化修饰之外,发生在天冬氨酸和谷氨酸侧链的甲基化修饰亦被认为具有非常重要的的调节功能。然而,能引起蛋白质体外甲基化(in vitro methylation)修饰的甲醇正被广泛地使用。本研究建立了一种蛋白质体外甲基化的质谱检测方法,并将其应用于牛血清白蛋白、鸡卵清白蛋白以及20S蛋白酶体复合体的体外甲基化修饰位点筛选。该质谱检测方法主要利用了峰标签技术(4P tag)以及质谱数据人工筛选(manual inspection of spectral data)方法。本研究旨在建立一种能有效地筛选蛋白质体外甲基化的质谱方法,揭示体外甲基化修饰的特点和反应特征,提出建议以尽量避免蛋白质体外甲基化修饰对体内甲基化(in vivo methylation)修饰研究的干扰,为今后蛋白质体内甲基化研究提供一定的参考价值。更多还原

【Abstract】 The 20S core particle (CP) is composed of 28 subunits arranged in four stacked heptameric rings (a7(37(37a7) forming a symmetrical barrel-shaped structure. Typically, in previous reports, the purification of CP mainly relied on the antibody, liquid chromatography and affinity tag-based strategies. In this study, we report a relatively simple, economical and effective protocol for proteomic analyses of CP, which only combines differential centrifugations with native-PAGE. Furthermore, it is compatible with both scaled-up purification from erythrocytes and scaled-down purification from low to around 3.0×10’pancreatic cancer cells, SW1990. This protocol was also applied for CP isolation from different species, such as yeast and mouse liver. In addition, a direct three-dimensional gel electrophoresis (d3-DE) approach that omits the interval procedure between native-PAGE and IEF/SDS-PAGE was developed. The results obtained in this study show that this protocol has a valuable potential for the studies of CP and other protein complexes. It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were seventeen proteolytic peptides with twenty modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior and characteristics of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, thus complementary confirmation for in vivo methylation, as well as the precautious guide for the use of methanol in proteomic and in vivo methylation studies.更多还原