2D PROTEIN ANALYSIS USING MICROCHIP MASS SPECTROMETRY

描述（改编自申请人的摘要）：目标是开发基于微芯片的IEF，并结合原位引入基质溶液，提供可用于直接MALDI-MS分析的蛋白质/基质混合物。IEF是根据等电点的差异来分离蛋白质的。一旦在三维（3-D） MEMS的下微通道中聚焦完成，将矩阵解决方案引入到上流道中。上流道通过活动隔板与下微流道垂直隔离。该板通过片外压电元件驱动，迫使板横向滑动，以在通道之间形成狭窄的开口。这种位移允许基质溶液与底层聚焦的蛋白质带直接混合，同时避免了对蛋白质纵向分辨率的干扰。基质晶体内的蛋白质可以使用MALDI-MS进行表征，具有极高的灵敏度、宽质量范围和对杂质的高耐受性。与二维分析系统类似，对三维MEMS的制造和微通道等电聚焦性能的基础研究为第一种分离机制奠定了基础。对原位引入基质溶液的基本了解和在各种分离添加剂存在下对MALDI-MS性能的评价是第二个分离维度。芯片上引入的矩阵溶液以自动化的方式消除了动员和电印迹蛋白质的需要，如在目前的毛细管和凝胶电泳分离。基于从本研究中获得的经验，在小型IEF阵列中同时分离分析蛋白并结合MALDI-MS分析，可以实现高通量蛋白质表达监测，包括翻译后修饰及其在不同生理和病理生理条件下的变化。由此产生的技术也将使研究人员能够接近人类基因组计划中相应蛋白质组的高通量分析。

DESCRIPTION (Adapted from the applicant's abstract): The goal is to develop microchip-based IEF coupled with in situ introduction of matrix solution, providing the protein/matrix mixtures which are amenable to direct MALDI-MS analysis. IEF separates proteins on the basis of their differences in isoelectric point. Once the focusing is complete in the lower microchannel of a 3-dimensional (3-D) MEMS, a matrix solution is introduced into the upper flow channel. The upper flow channel is vertically isolated from the lower microchannel by a movable partition plate. This plate is actuated through an off-chip piezoelectric element, forcing the plate to slide laterally for the creation of a narrow opening between the channels. This displacement permits direct mixing of matrix solution with the underlying focused protein bands, while avoiding disturbance in the longitudinal resolution of proteins. The proteins within the matrix crystals can be characterized using MALDI-MS with extremely high sensitivity, wide mass range, and high tolerance to impurities. In analogy to a 2-D analysis system, fundamental studies on the fabrication of 3-D MEMS and the performance of isoelectric focusing in the microchannel establish the basis for the first separation mechanism. Basic understanding of in situ introduction of matrix solution and the evaluation of MALDI-MS performance in the presence of various separation additives serve as the second separation dimension. The on-chip introduction of matrix solution in an automated fashion obviates the need to mobilize and electroblot proteins as in the current capillary and gel based electrophoretic separations. Building on experience gained from this proposed research, the simultaneous separation of analyte proteins in the array of miniaturized IEF together with MALDI-MS analysis may allow high throughput monitoring of protein expression, including posttranslational modifications and their changes under different physiological and pathophysiological conditions. The resulting technology will also enable the investigators to approach the high throughput analysis of the corresponding proteomes from the Human Genome Project.