2D PROTEIN ANALYSIS USING MICROCHIP MASS SPECTROMETRY

使用微芯片质谱法进行 2D 蛋白质分析

• 批准号: 2830132
• 负责人: CHENG S LEE
• 金额: $ 10.64万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2830132
• 关键词: bioimaging /biomedical imaging; biomedical equipment development; electrofocusing; matrix assisted laser desorption ionization; protein structure

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.

描述（改编自申请人的摘要）：目标是开发基于微芯片的IEF，并原位引入基质溶液，提供适合直接MALDI-MS分析的蛋白质/基质混合物。 IEF根据蛋白质等电点的差异分离蛋白质。 一旦在三维（3-D）MEMS的下部微通道中完成聚焦，就将基质溶液引入上部流动通道中。 上部流动通道通过可移动隔板与下部微通道垂直隔离。 该板通过片外压电元件致动，迫使板横向滑动以在通道之间形成窄开口。 这种位移允许基质溶液与下面的聚焦蛋白质条带直接混合，同时避免蛋白质纵向分辨率的干扰。基质晶体中的蛋白质可以使用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。与2-D分析系统类似，对3-D MEMS的制造和微通道中等电聚焦的性能的基础研究为第一分离机制建立了基础。 对基质溶液原位引入的基本理解和在各种分离添加剂存在下的MALDI-MS性能的评价作为第二分离维度。 以自动化方式在芯片上引入基质溶液避免了如在当前基于毛细管和凝胶的电泳分离中对动员和电印迹蛋白质的需要。 从这项拟议的研究中获得的经验的基础上，同时分离的分析物蛋白质的阵列中的小型化IEF连同MALDI-MS分析可以允许高通量监测蛋白质的表达，包括翻译后修饰及其在不同的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的相应蛋白质组进行高通量分析。