研究生基因组学材料.ppt

三、蛋白质组学 蛋白质组：一个基因组、一个细胞或组织、或一种生物体所表达的全部蛋白质 蛋白质组学：研究蛋白质组或应用大规模蛋白质分离和识别技术研究蛋白质组的一们学科 研究内容 技术手段 蛋白质结构及转录后修饰 蛋白质差异比较 蛋白质之间的相互作用 双向电泳 质谱 计算机图象分析/大规模数据处理 研究内容 蛋白质结构及转录后修饰 蛋白质差异比较 蛋白质之间的相互作用 技术手段 酵母/细胞双杂交 亲和层析 免疫沉淀 蛋白交联 激光共聚焦 从双向电泳到质谱的研究流程 蛋白酶解 蛋白酶解片段 蛋白点 双向电泳 凝胶成像 样品 MALDI-TOF 毛细管电泳 或HPLC N- 氨基酸序列测定 数据分析 LC/MS/MS 切胶 Theoretical pI and Mr map 6,216 yeast cell proteins From: Wildgruber et al. Electrophoresis 21 (2000) 2610-2616. pI (theoretical) Mr / kDa Working range for 2DE Note: protein abundance and hydrophobicity are not specifically highlighted in this picture 1,484 identified so far (Nat.Biotechnol. 17,676 and 19, 242) 商品化的多维色谱-质谱联用仪 IEF/SDS-LC/MS/MS是目前的新思路 * 說明文字 * From: Wildgruber R, Harder A, Obermaier C, Boguth G, Weiss W, Fey SJ, Larsen PM, G?rg A. Towards higher resolution: Two-dimensional electrophoresis of Saccharamyces cerevisiae proteins using overlapping narrow immobilized pH gradients. Electrophoresis. 21 (2000) 2610-2616. The theoretically calculated pIs from the open reading frames of Saccharomyces cerevisiae: More basic spots should be expected than acidic individuals.This picture implies that most theoretical proteins can be displayed in the orange frame. However, this map gives no indication if the proteins are high or low abundant, hydrophilic or hydrophobic. In reality less proteins will be visible on the gel. There is a potential chance that some of the missing proteins can be identified using LC based methods. The proteins below pH 6 are mostly enzymes and structural proteins. Between pH 5 and 7 there are not very many proteins expected, because proteins with pIs close to the pH of the medium have low solubility. The proteins with pIs over pH 7 are mostly present in low copy numbers; they are mainly regulatory proteins, with their basic pIs they can bind the acidic DNA much better. Demands on 2-D electrophoresis methodology: Stable basic gradients wide gradients from pH 3.5 to 13 high loading capacity to detect the proteins with low copy n