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学成像平台，以从复杂的生物样本中识别和表征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。该技术结合了凝胶电泳的卓越、高分辨率能力和质谱仪(MS)的灵敏度和高质量准确度。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的第一维)中成像，随后将采用凝胶范围的化学和酶方法以及MALDI-MS/MS进一步询问以提供序列、鉴定和翻译后/转录修饰的信息。我们的MS成像方法与其他自上而下的蛋白质组学和质谱学方法的不同之处在于：(1)我们寻求跟踪和阐明生物学家用于监测离散蛋白质异构体的存在、数量、酶活性和相互作用的分离形式中的修饰蛋白质，(2)我们寻求从仅有亚微克量的复杂样本中分析完整的蛋白质，以及(3)我们寻求将完整的蛋白质分析扩展到膜蛋白和小的开放阅读框架产品。“虚拟2D Gel/MS”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学可视化，将“自上而下”和“自下而上”蛋白质组学的优点联系起来。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，将动态蛋白质修改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。 公共卫生相关性：我们建议开发一种高通量技术来分析蛋白质状态，包括修饰，以及膜蛋白质和小分子质量蛋白质。目前的方法存在局限性，通常会阻止生物学家获取有关蛋白质形式的完整信息；这项技术将提醒他们缺乏但必要的信息。关于蛋白质形式以及它们在受伤或疾病后如何改变的更详细的信息可以获得。

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. PUBLIC HEALTH RELEVANCE: We propose to develop a high-throughput technology to profile protein status, including modifications, as well as membrane proteins and small molecular mass proteins. Current methods have limitations that generally prevent biologists from accessing complete information about protein forms; this technology would alert them to absent, but necessary information. More detailed information about protein forms and how they could be altered after injury or by disease can be obtained.