Novel Technologies for Protein Analysis

摘要 我的实验室的主要重点是开发蛋白质分析的新技术，特别是 围绕着蛋白质的概念。蛋白形式，每种蛋白形式都包含以下物质的独特组合： 氨基酸序列和翻译后修饰（PTMs）是细胞的主要分子效应物， 功能蛋白质型之间的细微序列和PTM差异可以完全改变它们的功能， 活动我们认为，对生物系统进行全面的蛋白质组水平分析， 了解它们的功能，包括细胞内的单个通路和网络，等等。 在全球范围内，破译控制细胞反应的系统生物学水平的动力学和相互作用。为 例如，我们的一个项目是阐明特定核酸序列和细胞之间的相互作用。 与DNA或RNA分子结合的蛋白形式。然而，今天的全球蛋白质合成技术 复杂系统的分析还处于起步阶段，这既是一个巨大的挑战，也是一个巨大的机遇。我们寻求 开发新的策略，用于复杂系统中的综合蛋白质型鉴定和定量。 我们设想结合来自多个数据流的信息，如转录组数据（以揭示剪接 形式和遗传变异），自下而上的蛋白质组学数据（揭示和定位PTM），自上而下的蛋白质组学 数据（为蛋白质型鉴定提供序列标签）和完整质量测量（鉴定和 使用来自所有其它数据流的信息来量化蛋白质型）。具体项目将开发 以下内容：（1）用于构建样品特异性蛋白质组数据库的强大工具;（2）用于 PTMs的发现和定位;（3）改进样品制备、分离和质谱分析 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化，以最大限度地提高蛋白质型鉴定;和（5）可视化工具， 显示相关蛋白质型之间的连接和变化的蛋白质型家族。我们将整合这些 方法和数据流，以及强大的开源软件和附带的协议， 这些能力广泛可用，使各地的研究人员能够更深入地了解 他们的生物系统的功能。我们将把我们的创新工具应用于许多尖端项目， 众多的合作者，因为技术开发在相关的背景下是最有意义的 生物学研究，因为它将增加科学家对蛋白质型分析的采用， 更广泛的生物医学社区。

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.