Mass Spectrometry Imaging of Protein Gels

描述（由申请人提供）：我们建议开发一个高通量，集成的自上而下的质谱成像平台，以鉴定和表征复杂生物样品中的蛋白质异构体和表达谱，并阐明蛋白质修饰对蛋白质功能和相互作用的影响。该技术将凝胶电泳的高分辨率功能与质谱（MS）的灵敏度和高质量精度相结合。通过等电聚焦凝胶电泳分离的完整蛋白质将通过自动、高速基质辅助激光解吸/电离（MALDI） MS从固定化pH梯度（IPG）聚丙烯酰胺凝胶（经典2D-PAGE的第一个维度）上成像，随后将采用全凝胶化学和酶方法，并通过MALDI-MS/MS进一步查询，以提供序列、鉴定和翻译后/转录修饰的信息。我们的质谱成像方法与其他自上而下的蛋白质组学和质谱方法的区别在于：(1)我们寻求跟踪和阐明生物学家用于监测离散蛋白质同种异构体的存在、数量、酶活性和相互作用的分离格式的修饰蛋白质；(2)我们寻求从亚微克数量的复杂样品中分析完整的蛋白质；(3)我们寻求将完整蛋白分析扩展到膜蛋白和小的开放阅读框产品。“虚拟2D凝胶/质谱”平台可能会在不到30分钟的时间内实现超高分辨率分离的质谱可视化，将“自上而下”和“自下而上”蛋白质组学的优势联系起来。我们的愿景是，这项技术将为生物学家和质谱学家提供几乎前所未有的能力，将动态蛋白质修饰与特定疾病或细胞反应联系起来，并且该平台可以广泛部署。

DESCRIPTION (provided by applicant): We propose to develop a high-throughput, integrated top-down mass spectrometry imaging platform to identify and characterize protein isoforms and expression profiles from complex biological samples and to elucidate effects of protein modifications on protein function and interactions. The technology combines the premier, high resolution capabilities of gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing gel electrophoresis will be imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by automated, high-speed matrix-assisted laser desorption/ionization (MALDI) MS. This will be followed by gel-wide chemical and enzymatic methods and further interrogation by MALDI-MS/MS to provide information on sequence, identification, and post-translational/transcriptional modifications. Our MS imaging approach is distinguished from other top-down proteomic and mass spectrometry methods in that (1) we seek to track and to elucidate modified proteins from separation formats employed by biologists to monitor presence, amount, enzymatic activity, and interactions of discrete protein isoforms, (2) we seek to profile intact proteins from complex samples that are available in only sub-microgram quantities, and (3) we seek to extend intact protein analyses to membrane proteins and small open reading frame products. The "Virtual 2D gel/MS" platform may potentially lead to mass spectrometric visualization of ultrahigh resolution separations in less than 30 minutes, linking the benefits of "top-down" and "bottom-up" proteomics. Our vision is that the technology will provide biologists and mass spectrometrists with nearly unprecedented capability to link dynamic protein modifications to specific disease or cellular responses, and that the platform can be widely deployed.