Novel Technologies for Protein Analysis

摘要 我的实验室的主要重点是开发新的蛋白质分析技术，特别是 以蛋白质形式的概念为中心。蛋白质组，其中每一种都由独特的 氨基酸序列和翻译后修饰(PTM)是细胞的主要分子效应物 功能。蛋白质形式之间微妙的序列和PTM差异可以完全改变它们的功能和 活动。我们认为全面的蛋白质形式水平的生物系统分析是绝对必要的 了解它们的功能，包括单个通路和细胞内运行的网络，等等 在全球范围内，破译控制细胞反应的系统-生物水平的动力学和相互作用。为 例如，我们的项目之一是阐明特定的核酸序列和 蛋白质形式结合在这些DNA或RNA分子上。然而，当今全球蛋白质形式的技术 复杂系统的分析还处于初级阶段，这既是一个巨大的挑战，也是一个巨大的机遇。我们在寻找 为复杂系统中蛋白质形式的综合鉴定和量化开发新的策略。 我们设想组合来自多个数据流的信息，例如转录数据(以揭示剪接 形式和遗传变异)、自下而上的蛋白质组学数据(以揭示和定位PTMS)、自上而下的蛋白质组学 数据(为蛋白质形式鉴定提供序列标签)和完整质量测量(鉴定和 使用来自所有其他数据流的信息来量化蛋白质形式)。具体的项目将开发 以下内容：(1)用于构建特定样本蛋白质形式数据库的强大工具；(2)新战略 PTMS的发现和定位；(3)改进了样品的制备、分离和质谱学 完整蛋白质的方法；(4)利用完整质量测量和 选择自上而下的片段以最大限度地鉴定蛋白质形式；以及(5)可视化工具 蛋白形式家族，显示相关蛋白形式之间的联系和变化。我们将把这些整合在一起 方法和数据流，以及功能强大的开源软件和附带的协议 这些功能广泛可用，使世界各地的研究人员能够更深入地了解 他们的生物系统的运作。我们将把我们的创新工具应用到许多尖端项目中 众多合作者，既是因为技术开发在相关背景下最有意义 生物研究，因为它将增加蛋白质形式分析在科学家中的应用 更广泛的生物医学界。

Abstract The primary focus of my laboratory is the development of novel technologies for protein analysis, specifically centered around the concept of the proteoform. Proteoforms, each of which comprises a unique combination of amino acid sequence and post-translational modifications (PTMs), are the primary molecular effectors of cell function. Subtle sequence and PTM differences between proteoforms can completely alter their function and activity. We see comprehensive proteoform-level analysis of biological systems as absolutely essential to understanding their function, for both individual pathways and networks operative within cells, and more globally, to decipher the systems-biology-level dynamics and interactions that control cellular response. For example, one of our projects is to elucidate the interactome between specific nucleic acid sequences and the proteoforms bound to those DNA or RNA molecules. However, today's technology for global proteoform analysis in complex systems is in its infancy, offering both a great challenge and a great opportunity. We seek to develop novel strategies for comprehensive proteoform identification and quantification in complex systems. We envision combining information from multiple data streams, such as transcriptomic data (to reveal splice forms and genetic variation), bottom-up proteomics data (to reveal and localize PTMs), top-down proteomics data (to provide sequence tags for proteoform identifications), and intact mass measurements (to identify and quantify proteoforms, using information from all of the other data streams). Specific projects will develop the following: (1) robust tools for the construction of sample-specific proteoform databases; (2) new strategies for the discovery and localization of PTMs; (3) improved sample preparation, separation, and mass spectrometry methods for intact proteins; (4) synergistic approaches that utilize both intact mass measurements and selected top-down fragmentations to maximize proteoform identifications; and (5) visualization tools for proteoform families that show connections and changes between related proteoforms. We will integrate these methods and data streams together with powerful open-source software and accompanying protocols to make these capabilities widely available, enabling researchers everywhere to gain a deeper understanding of the functioning of their biological systems. We will apply our innovative tools to many cutting-edge projects with numerous collaborators, both because technology development is most meaningful in the context of relevant biological studies and because it will increase the adoption of proteoform analysis among scientists in the broader biomedical community.