Mass Spectrometry Imaging of Protein Gels

蛋白质凝胶的质谱成像

• 批准号: 8657073
• 负责人: Joseph A Loo
• 金额: $ 29.52万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-23 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8657073
• 关键词: Algae; Bacillus subtilis; Benchmarking; Biological; Biological Assay; Calibration; Cataloging; Catalogs; Cells; Chemicals; Chlamydomonas reinhardtii; Complex; Computer software; Data; Data Analyses; Deposition; Detection; Dimensions; Disease; Environment; Evaluation; Gel; Glycoproteins; Goals; Image; Imagery; Injury; Ions; Isoelectric Focusing; Isoelectric Point; Lead; Link; Mass Spectrum Analysis; Measurement; Measures; Membrane Proteins; Methods; Modification; Molecular Profiling; Molecular Weight; Monitor; Open Reading Frames; Peptide N-glycohydrolase F; Peptides; Phosphorylation; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Proteome; Proteomics; Resolution; Sampling; Slice; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Spottings; Staining method; Stains; Techniques; Technology; Vacuum; Vision; base; computerized data processing; data acquisition; gel electrophoresis; glycosylation; high throughput technology; instrument; molecular mass; pH gradient; polyacrylamide gels; prevent; protein complex; protein expression; protein function; protein profiling; response; virtual

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.

描述（由申请人提供）：我们建议开发一种高通量、集成的自上而下的质谱成像平台，以识别和表征复杂生物样品中的蛋白质亚型和表达谱，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一流高分辨率功能与质谱 (MS) 的灵敏度和高质量准确度相结合。通过等电聚焦凝胶电泳分离的完整蛋白质将通过自动化高速基质辅助激光解吸/电离 (MALDI) MS 从固定 pH 梯度 (IPG) 聚丙烯酰胺凝胶（经典 2D-PAGE 的第一维）中成像。随后将进行全凝胶化学和酶法，并通过 MALDI-MS/MS 进一步询问，以提供有关序列、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方法的区别在于 (1) 我们寻求从生物学家使用的分离格式中跟踪和阐明修饰的蛋白质，以监测离散蛋白质亚型的存在、数量、酶活性和相互作用，(2) 我们寻求从仅亚微克数量的复杂样品中分析完整的蛋白质，(3) 我们寻求扩展 对膜蛋白和小开放阅读框产品的完整蛋白质分析。 “虚拟 2D 凝胶/MS”平台有可能在 30 分钟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，该技术将为生物学家和质谱学家提供近乎前所未有的能力，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。