【作者】 张丽军；

【导师】 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 细胞质膜（PM）是细胞与环境直接接触的界面。镶嵌或连接于其中的蛋白质作为细胞的“门铃”与“门户”，是现有的70％的药物的靶标，因此细胞质膜蛋白质组的研究具有重要的理论与实际应用意义。然而，由于质膜蛋白质具有难溶、低丰度以及组成复杂等特点，是一项具有极大挑战性的研究。 首先，本文以鼠肝为材料，对质膜蛋白质组的研究进行了一系列的方法摸索。肝脏在人体的生命活动中具有非常重要的功能，是一个复杂性仅次于大脑的器官。分析其质膜蛋白质组将有助于对肝脏的认识和肝病的治疗。我们用蔗糖密度梯度离心法纯化质膜，优化出适合2DE分离膜蛋白的裂解液（含4％w／v CHAPS和1％NP-40等）和初步摸索出整合膜蛋白质的富集方法（氯仿和甲醇抽提，Na₂CO₃处理）。对用2DE和SDS-PAGE分离的蛋白质进行质谱鉴定，一共鉴定了175个非冗余蛋白质。其中9％的蛋白质功能未知，生物学分析发现四个未知蛋白质可能与肌肉营养不良等疾病有关。其次，为了鉴定低丰度、强疏水性整合质膜蛋白质，我们用Na₂CO₃处理，氯仿与甲醇抽提，以及质膜微区分离策略来富集这些蛋白质。通过2DE／1DE-MS／MS的技术路线分析，一共鉴定了457个非冗余蛋白质，其中23％的带有一个或一个以上跨膜区（TM）。在这457个蛋白质中，有53个与细胞通信相关。对这些鉴定的蛋白质进行emPAI分析，发现有50％的蛋白质的浓度低于0.1M，其中12％的蛋白质浓度比arginase 1和actin的低100倍。对鉴定蛋白质的理化性质分析，发现Na₂CO₃处理有利于碱性蛋白质研究，氯仿与甲醇有利于多跨膜区蛋白质研究，微区策略有利于低丰度蛋白质研究。再有，为了提高质膜的纯度，采用抗体磁珠免疫亲和纯化方法来纯化质膜。通过对4种富集方法的比较，找到了最佳的纯化方法。与密度梯度离心的结果相比，这种方法能将质膜的纯度提高3倍，线粒体的污染减少了2倍，内质网的污染几乎被去除。随后，我们优化了初质膜、磁珠以及抗体的用量，得到了适合蛋白质组研究的较少用量。对纯化的质膜蛋白质进行SDS-PAGE分析，质谱鉴定。更多还原

【Abstract】 Plasma membrane （PM） constitutes the interface between cells and their external environment. The proteins embedded in this membrane work as the "doorbells" and "doorways" of the cells, and are the targets of about 70% of all known drugs. For these reasons, proteomic analysis of the plasma membrane is very important for theoretical and applied cytology and pharmacology. However, due to the low abundance, high hydrophobicity and the evidently complexity of PM proteins, it is very challenging to study.First, we used mouse/rat liver as material, and looked for the methods for PM proteome research. The liver is one of the most important organs in the body, probably second only to the brain in organ complexity. Therefore, proteomic analysis of liver PM will be helpful to understand these physiology activities and treat liver disease. We isolated PM from mouse liver by sucrose density gradient centrifugation. The optimized lysis buffer containing 4% CHAPS and 1% NP-40 was obtained. The methods for integral membrane protein enrichmentwere analyzed and better methods----chloroform/methanolextraction and sodium carbonate treatment were found. The extracted PM proteins were separated by 2DE and SDS-PAGE, and submitted to MS identification. In all, 175 non-redundant proteins were identified, of which 9% proteins with unknown function. Through prediction, four unknown proteins were found to be related with limb-girdle muscular dystrophy 2B, and so on. Second, in order to characterize low copy integral membrane proteins. Integral plasma membrane proteins were enriched through the treatment with 0. 1M Na₂CO₃, chloroform/methanol更多还原