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，包括细胞裂解物和血清，含有数百甚至数千种蛋白质，2-D凝胶电泳分离（2D-PAGE）是用于分离和分离单个蛋白质以进行分析的标准方法。除了用于鉴定单个蛋白质外，它还用于监测特定蛋白质或蛋白质的量的变化，作为组织中疾病发作的指示，或用于研究疾病如何发展以及病理如何受其环境和治疗方法的影响。我们将开发与标准2D-PAGE方法相当的毛细管，与2D-PAGE相比，该方法提供了几项改进，包括自动化、单个蛋白质的定量测量、显著减少的运行时间、用于蛋白质鉴定的可再现的大小和pI测量，以及用于将感兴趣的特定蛋白质存款到MALDI板上用于TOP质谱鉴定的接口。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）耦合的方案，所述毛细管阵列具有用于类似于2D-PAGE的自动化二维电泳的荧光检测，以及将毛细管阵列的输出耦合到MALDI板以用于“离线”蛋白质鉴定的方案。在第一阶段，将开发和测试两种耦合方案。此外，将研究样品的预分级以提高灵敏度和分离效率。这对于分析全细胞样本、血清和其他体液以用于癌性和癌前组织的临床诊断和研究来说将是一个重大的进步。MALDI接口的毛细管阵列将是这种能力的第一次演示。

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.