An Automated 2D-CAE System Interfaced to MALDI MS

描述（由申请人提供）：对于复杂的蛋白质混合物，包括细胞裂解液和血清，含有数百甚至数千种蛋白质，2d凝胶电泳分离（2D-PAGE）是用于分离和分离单个蛋白质进行分析的标准方法。除了用于识别单个蛋白质之外，它还用于监测特定蛋白质或蛋白质数量的变化，作为组织中疾病发病的指示，或用于研究疾病如何发展以及病理如何受到其环境和治疗方法的影响。我们将开发一种相当于标准2D-PAGE方法的毛细管，与2D-PAGE相比，它提供了一些改进，包括自动化，单个蛋白质的定量测量，显著减少的运行时间，可重复的尺寸和pI测量蛋白质鉴定，以及将感兴趣的特定蛋白质沉积到MALDI板上进行TOP质谱鉴定的界面。创新包括将玻璃微流控芯片通道中的等电聚焦（CIEF）与毛细管阵列中的凝胶电泳（CGE）耦合在一起的方案，该方案具有类似于2D-PAGE的自动二维电泳的荧光检测，以及将毛细管阵列的输出耦合到MALDI板以进行“离线”蛋白质鉴定的方案。在第一阶段，将开发和测试这两种耦合方案。此外，还将研究样品的预分馏以提高灵敏度和分离效率。这将是对全细胞样本、血清和其他体液进行临床诊断和癌症及癌前组织研究的重大进展。与毛细管阵列的MALDI接口将是这种能力的第一个演示。

DESCRIPTION (provided by applicant): For complex protein mixtures, including cell lysates and serum, containing hundreds and even thousands of proteins, 2-D gel-electrophoresis separation (2D-PAGE) is the standard method used to separate and isolate individual proteins for analysis. In addition to being useful for identifying individual proteins, it is also used to monitor changes in the amount of a specific protein or proteins as an indication of the onset of a disease in tissue or to study how disease develops and how pathology is affected by its environment and by methods to treat it. We will develop a capillary equivalent to the standard 2D-PAGE method that offers several improvements compared to 2D-PAGE, including automation, quantitative measurement of individual proteins, dramatically decreased run time, reproducible size and pI measurement for protein identification, and an interface to deposit specific proteins of interest onto a MALDI plate for TOP mass spectrometry identification. Innovations include a scheme to couple isoelectric focusing (CIEF) in a glass micro-fluidic chip channel with gel electrophoresis (CGE) in a capillary array with fluorescence detection for automated two-dimensional electrophoresis analogous to 2D-PAGE and a scheme to couple the output of a capillary array to a MALDI plate for "off-line" protein identification. In Phase I, both coupling schemes will be developed and tested. In addition, pre-fractionation of samples to improve sensitivity and separation efficiency will be investigated. This will be a significant advance for the analysis of whole cell samples, serum, and other bodily fluids for clinical diagnosis and research studies of cancerous and pre-cancerous tissue. A MALDI interface to a capillary array will be the first demonstration of this capability.