Intact Proteoform Identification and Quantification

 描述(申请人提供)：生物系统中的关键蛋白质成分是完整的蛋白质形式，即由基因组以各种剪接形式产生的不同形式的蛋白质，并带有无数显著影响其功能的翻译后修饰。遗憾的是，今天的自下而上的蛋白质组学技术未能向生物学家提供关键的蛋白质水平信息，这些技术识别和量化来自蛋白质的多肽，而不是蛋白质本身。我们在这里通过开发一种新的蛋白质组学分析范式来解决这个问题，该范式使用完整的质量和赖氨酸计数来识别和量化生物样本中存在的蛋白质形式。我们的战略集成了最先进的蛋白质组和基因组技术。我们结合了新一代高分辨率质谱仪和一种新的同位素标记策略，为每种蛋白质形式提供其准确的质量、其赖氨酸数量和其相对丰度。对同一样本的平行RNA-Seq分析将提供转录数据，以构建紧密聚焦的样本特异性蛋白质形式数据库。质谱学数据与蛋白质形态数据库的比较将揭示样品中存在的蛋白质形态特征，同位素标记策略将提供相对丰度。这些信息将构成一种革命性的生物学新工具。

 DESCRIPTION (provided by applicant): The critical protein actors in biological systems are the intact proteoforms, namely the different forms of proteins produced from the genome in a variety of splice forms and adorned with a myriad of post-translational modifications that dramatically affect their function. Lamentably, today's bottom-up proteomic technologies, which identify and quantify peptides derived from proteins, rather than the proteins themselves, fail to deliver crucial protein-level information to biologists. We address this problem here, by developing a new paradigm for proteomic analyses, which uses intact mass and lysine count to identify and quantify the proteoforms present in a biological sample. Our strategy integrates state-of-the-art proteomic and genomic technologies. We couple a new generation of high resolution mass spectrometers and a novel isotopic tagging strategy to yield for each proteoform its accurate mass, its number of lysines, and its relative abundance. Parallel RNA-Seq analyses on the same sample will provide transcriptomic data to construct a tightly focused sample-specific proteoform database. Comparison of the mass spectrometric data with the proteoform database will reveal the proteoform identities present in the sample, and the isotopic tagging strategy will provide relative abundance. This information will comprise a revolutionary new tool for biology.