毛细管电泳激光诱导荧光检测蛋白质-生物化学与分子生物学专业毕业论文.docxVIP

华中科技大学硕士学位论文 华 中 科 技 大 学 硕 士 学 位 论 文 I I 摘 要 随着后基因组时代的到来，生物研究的重心逐渐从 DNA、RNA 等遗传物质转移 到蛋白质等功能物质上来。蛋白质组学研究，尤其是单细胞蛋白质组研究对当前的 生物大分子分离、分析技术提出了新的要求。 毛细管电泳曾以其快速高效的特点极大的推动了人类基因组计划的完成，并大 大降低了开销。而在蛋白质分离分析领域，毛细管电泳又以其纳升级的进样量，纳 摩尔级的检测极限和极高的分辨率越来越发挥出难以取代的作用。 本文主要研究了基于毛细管电泳的高灵敏度、高分辨率的蛋白质分离、激光诱 导荧光检测系统的构建以及标准蛋白质样品毛细管电泳分离条件的摸索。本系统采 用毛细管无胶筛分电泳模式，蛋白质样品在进样之前用 FQ 荧光发生试剂进行衍生， 得到具有荧光特性的蛋白质-FQ 衍生物。使用 3 万伏高压电源进行电泳分离、蛋白质 -FQ 衍生物在波长为 488 纳米的氩离子激光诱导下产生荧光，荧光信号再通过滤镜组 被光电倍增管收集放大，滤波，通过数据采集程序采集，得到三种标准蛋白质的电 泳图谱。通过对图谱中分离度、峰高和灵敏度等参数的分析，改变缓冲液 pH、分离 场强、筛分介质浓度和 SDS 浓度等一系列分离相关的条件，获得较好的分离效果， 基本达到高灵敏度、高分辨率的要求。 关键词：毛细管电泳 蛋白质 激光诱导荧光 II II Abstract High-throughput sequencing and functional genomics technologies have given us a draft of human genome sequence. The focus is now shifting to downstream functional molecules like microRNA, snRNA and protein. Proteomic research, especially single cell proteomics ask for new ananylis technologies with higher sensitivity and better resolution. Capillary electrophoresis (CE) played a critical role in Human Genome Project, accelerating the progress, minimizing the cost. It also has much advantage on Proteomics with injection volume about several nanoliters, low detection limit (nanomole/L) and ultra high resolution. In this thesis, we investigated a protein separation and detection system based on CE and laser induced fluorescence (LIF) detection. Three standard proteins were successfully separated and detected in our home-built system. We used the capillary sieving electrophoresis (CSE) mode for protein separation. Before electrophoresis, proteins reacted with FQ to acquire fluorescent characteristic to enable detection by LIF after separation. CE was performed in a inner wall-coated capillary under a high-voltage source capable of providing maximum voltage up to 30kV. A 488nm laser beam was focused in the middle of the detection window on the capillary to excite sample zones passing through. Fluorescence was collected by an objective in an IX71 microscopy system, co