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

用于癌症基质蛋白二维组织定位和更深入蛋白质组测序的酶工具

基本信息

批准号：
9794266
负责人：
Peggi M Angel
金额：
$ 24.01万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9794266
关键词：
Address Advanced Malignant Neoplasm Amino Acid Sequence Amino Acids Area Basic Science Biochemical COL1A1 gene COL1A2 gene Caliber Cancer Biology Cells Cirrhosis Cleaved cell Clinical Communities Data Detection Development Diagnosis Environment Enzymes Exploratory/Developmental Grant Fibroblasts Formalin Freezing Future Gelatinase B Genes Growth Heterogeneity Image Interstitial Collagenase Isotope Labeling Left Literature Malignant Neoplasms Mass Spectrum Analysis Matrix Metalloproteinases Measurement Methods Microscopy Neoplasm Metastasis Noise Paraffin Embedding Pathologic Pathology Peptide Hydrolases Peptides Pharmaceutical Preparations Post-Translational Protein Processing Preparation Primary carcinoma of the liver cells Production Prognostic Marker Protein Isoforms Proteins Proteome Proteomics Protocols documentation Publishing Reaction Reagent Recombinants Reporting Research Research Personnel Role Sampling Signal Transduction Signaling Protein Solid Specificity Specimen Stromal Cells Technology Testing Thinness Tissue Microarray Tissue Model Tissue Preservation Tissues Trypsin Tumor Tissue Variant Work anticancer research cancer heterogeneity cell type cohort collagenase collagenase 3 commercialization crosslink deep sequencing diagnostic biomarker enzyme activity experience experimental study human tissue improved liver biopsy outcome forecast protein aminoacid sequence single cell proteins success tool tumor tumor microenvironment tumor progression type I collagen alpha 1

项目摘要

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.

由于缺乏工具，确定基质蛋白在癌症生物学中的作用一直很困难。当前的工具是定性的，无法报告蛋白质序列变化，这对于了解基础的方式至关重要蛋白质有助于或阻碍癌症的生长和转移。我们以前的工作表明细菌胶原酶可用于产生通过质谱法检测到的基质蛋白质组作为癌组织上的2D分布。我们仍然缺乏可以将重点放在靶蛋白上的工具，以详细研究序列变化和后翻译修改。我们建议使用基质金属蛋白酶（MMP）作为针对性的酶促工具检测癌组织微环境中基质蛋白序列。目标1，我们将发展并优化一套MMP，以分析肿瘤组织切片中基质序列。我们将生产并优化专门用于组织切片的2D固体底物的重组MMP。我们将整合使用当前定性工具的序列信息，扩大基质蛋白序列检测功能下到单个细胞小众。在AIM 2中，我们将专注于制定定量检测目标的标准来自组织微环境的基质蛋白。我们将针对小型但肝硬化的相关肝细胞癌的相关队列。我们的研究团队有一个独特的 MMP的生产，开发和未来商业化的专业知识的结合。提案具有变革性，因为它将允许检测固定临床标本的基质蛋白序列变化传统方法是不可能的。一个长期目标是显着改善社区的能够靶向肿瘤组织微环境中用于基础研究的基质蛋白，指导疗法，或预后或诊断生物标志物。