Enzymatic Tools for 2D Tissue Localized and Deeper Proteomic Sequencing of Cancer Stromal Proteins

由于缺乏工具，确定基质蛋白在癌症生物学中的作用仍然很困难。电流 工具是定性的，不能报告蛋白质序列变异，这对理解基质如何 蛋白质帮助或阻碍癌症生长和转移。我们以前的工作表明，细菌胶原酶 可用于产生基质蛋白质组，其通过质谱法检测为癌组织上的2D分布。 我们仍然缺乏工具，可以专注于目标蛋白质的序列变异和后的详细研究， 翻译修饰我们建议使用基质金属蛋白酶（MMPs）作为酶促工具， 检测癌组织微环境中的基质蛋白序列。目标1，我们将开发和 优化一套MMP用于分析肿瘤组织切片中的基质序列。我们将生产和 优化重组MMP，特别是用于组织切片的2D固体基质。我们将整合 利用当前的定性工具提供序列信息，扩展基质蛋白序列检测能力 到一个单细胞龛。在目标2中，我们将专注于制定定量检测目标的标准 从组织微环境中分离基质蛋白。我们将测试开发的技术对一个小的，但 肝硬化后出现肝细胞癌的相关队列。我们的研究团队有一个独特的 MMPs的生产，开发和未来商业化的专业知识的组合。该提案 因为它将允许从固定的临床样本中检测基质蛋白序列变异 这在传统方法中是不可能的。长远目标是大幅改善社区的 能够靶向肿瘤组织微环境中的基质蛋白，用于基础研究， 治疗或预后或诊断生物标志物。

Determining the role of stromal proteins in cancer biology has remained difficult due to a lack of tools. Current tools are qualitative and cannot report on protein sequence variation that is critical to understanding how stromal proteins help or impede cancer growth and metastasis. Our previous work has shown that bacterial collagenases can be used to produce stromal proteomes detected by mass spectrometry as 2D distributions on cancer tissues. We still lack tools that can focus down on target proteins for detailed studies of sequence variation and post- translational modification. We propose using matrix metalloproteinases (MMPs) as enzymatic tools for targeted detection of stromal protein sequences in the cancer tissue microenvironment. Aim 1, we will develop and optimize a suite of MMPs for analysis of stromal sequences in tumor tissue sections. We will produce and optimize recombinant MMPs specifically for use on 2D solid substrates of tissue sections. We will integrate sequence information with current qualitative tools, expanding capabilities for stromal protein sequence detection down to a single cell niche. In Aim 2, we will focus on developing standards for quantitative detection of target stromal proteins from the tissue microenvironment. We will test the developed technology against a small but relevant cohort of emergent hepatocellular carcinoma from cirrhosis. Our research team has a unique combination of expertise for the production, development, and future commercialization of MMPs. The proposal is transformative since it will allow detection of stromal protein sequence variation from fixed clinical specimens which is impossible with conventional methods. A long-term objective is to significantly improve the community's ability to target stromal proteins within the tumor tissue microenvironment for use in basic research, directing therapies, or prognostic or diagnostic biomarkers.