Spatially resolved characterization of proteoforms for functional proteomics

项目摘要/ABSTARCT 分化的细胞有不同的表观遗传标记模式，包括各种翻译后标记 组蛋白上的修饰(PTM)可能协同作用来控制转录程序。自表观遗传以来 在暴露于环境毒素后，标志通常会改变，并在疾病中扮演多种角色 发病机制，即在组织和细胞环境中测量组蛋白的能力是一个主要的分析目标和 挑战。基于质谱学(MS)的蛋白质组学是确定组蛋白改变的有力工具 多元化和无针对性的时尚。然而，传统的自下而上(即多肽级)MS不能提供 多个PTM的化学计量比和组合的完整表征，以及其他组合 变异的来源，这些变异共同构成了任何单个基因的一组蛋白质形式(即 蛋白质组)。自上而下(即蛋白质形式级别)MS通过省略蛋白质分解来解决这一挑战，从而 从而允许获得功能蛋白形式。然而，自上而下的MS存在灵敏度低和动态化的问题 由于分离和检测大丰度和低丰度蛋白质的挑战和繁琐的范围 获得高蛋白质组覆盖率所需的纯化步骤。这严重限制了我们分析小问题的能力 并使用自上而下的MS来生成更深层次所需的组织的蛋白质形态感知图像 了解人体器官在健康和疾病中的功能。我们最近开发出了纳米液滴 用于高灵敏自下而上蛋白质组学的样品制备(纳米POTS)，并将该方法扩展到组织 以100微米的空间分辨率成像。在此，我们建议开发和部署基于纳米POTS的自上而下 MS能够以接近单细胞分辨率的方式表征组织切片中的蛋白质形式。为了增加 为了实现从数千个细胞到近单个细胞的分辨率，我们将采用先进的MS成像(MSI)方法。 MSI数据将与通过微尺度自上而下的MS获得的全球蛋白质组学数据进行交叉参考 显微解剖的组织区域。UG3阶段的工作将集中在组蛋白和肾脏作为开发 平台，并利用微规模自上而下的LCMS、MSI和新颖的图像处理的独特组合 和可视化工具。在UH3阶段，我们将构建多个 组织类型和促进肾脏特定功能单元的多模式分子图谱 HubMAP联盟正在努力。成功完成这项研究将允许全面 组织和细胞中蛋白质形式的全谱的特征，从而解决了一个重要的和不足的- 研究的生物学领域和HuBMAP努力中的关键差距。

PROJECT SUMMARY/ABSTARCT Differentiated cells have distinctive patterns of epigenetic marks including various post-translational modifications (PTMs) on histones that may work in concert to control transcriptional programs. Since epigenetic marks are often altered following exposure to environmental toxins and play multiple roles in disease pathogenesis, the ability to measure histones in a tissue and cell context is a major analytical objective and challenge. Mass spectrometry (MS) based proteomics is a powerful tool for characterizing histone alterations in multiplexed and non-targeted fashion. However, conventional bottom-up (i.e. peptide-level) MS cannot provide complete characterization of the stoichiometry and combinations of multiple PTMs, and other combinatorial sources of variation, that collectively make up any single gene's set of proteoforms (i.e. functional units of a proteome). Top-down (i.e. proteoform-level) MS addresses this challenge by omitting the proteolysis and thus allowing access to the functional proteoforms. However, top-down MS suffers from low sensitivity and dynamic range due to challenges in separation and detection of large and low-abundance proteins and laborious purification steps required to achive high proteome coverage. This severely limits our ability to analyze small samples and employ top-down MS to generate proteoform-aware images of tissues required for a deeper understanding of human organ functioning in health and disease. We have recently developed nanodroplet sample preparation (nanoPOTS) for highly sensitive bottom-up proteomics and extended this approach to tissue imaging with 100 µm spatial resolution. Herein, we propose to develop and deploy nanoPOTS-based top-down MS to enable characterization of proteoforms in tissue sections with near single cell resolution. To increase the resolution from thousands of cells to near single cell, we will employ advanced MS imaging (MSI) approaches. MSI data will be cross-referenced with global proteomics data obtained via microscale top-down MS of microdissected tissue regions. The UG3 phase efforts will be focused on histones and kidney as a development platform and leverage a unique combination of microscale top-down LCMS, MSI and novel image processing and visualization tools. In the UH3 phase, we will construct comprehensive proteoform-specific maps of multiple tissue types and facilitate multimodal molecular mapping of specific functional units of the kidney by leveraging the HubMAP Consortium ongoing efforts. Successful completion of this research will allow for comprehensive characterization of the full spectrum of proteoforms in tissues and cells thus addressing an important and under- studied area of biology and critical gap in HuBMAP efforts.