2D PROTEIN ANALYSIS USING MICROCHIP MASS SPECTROMETRY

描述(摘自申请人的摘要)：目标是开发基于微芯片的离子交换检测器，并在原位引入基质溶液，提供能够直接进行MALDI-MS分析的蛋白质/基质混合物。IEF根据蛋白质等电点的不同来分离蛋白质。在三维MEMS的下部微通道中完成聚焦后，在上部流动通道中引入基质溶液。通过活动隔板将上流道与下微通道垂直隔离。这种板是通过片外压电元件驱动的，迫使板横向滑动，从而在通道之间形成一个狭窄的开口。这种置换允许基质溶液与潜在的聚焦蛋白质带直接混合，同时避免了对蛋白质纵向分辨率的干扰。基质晶体中的蛋白质可以用MALDI-MS进行表征，具有极高的灵敏度、宽的质量范围和对杂质的高耐受性。类似于二维分析系统，对三维MEMS的制作和微通道内等电聚焦性能的基础研究为第一种分离机制奠定了基础。第二个分离维度是对就地引入基质溶液的基本了解和在存在各种分离添加剂时对MALDI-MS性能的评价。芯片上以自动方式引入基质溶液，不需要像目前基于毛细管和凝胶的电泳分离那样动员和电印迹蛋白质。基于从这项拟议研究中获得的经验，在小型化IEF阵列中同时分离分析蛋白质并结合MALDI-MS分析可能允许高通量监测蛋白质表达，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。由此产生的技术还将使研究人员能够对人类基因组计划中相应的蛋白质组进行高通量分析。

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.