Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment

Q1 Agricultural and Biological Sciences

Current protocols in mouse biology Pub Date : 2020-09-08 DOI:10.1002/cpmo.83

Jason Yeung, Jeffrey Lamb, Jonathan R. Krieger, Mihaela Gadjeva, Jennifer Geddes-McAlister

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Abstract

Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Murine model of ocular infection

Basic Protocol 2: Murine model sample collection

Basic Protocol 3: Protein extraction from eye wash

Basic Protocol 4: Protein extraction from corneal tissue

Basic Protocol 5: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples

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小鼠眼组织和细胞外环境的定量蛋白质组学分析

基于质谱的蛋白质组学为检测和定量样品(包括细胞、组织、器官和上清液)中蛋白质丰度的变化提供了一种强大而可靠的方法。物理损伤或炎症可损害眼表，使细菌病原体定植，通常是铜绿假单胞菌，并形成生物膜。铜绿假单胞菌与感染部位的免疫系统之间的相互作用决定了宿主抵御细菌入侵和促进清除感染的能力。感染后眼组织的特征描述了宿主先天免疫反应的性质，特别是中性粒细胞相关蛋白的存在和丰富程度，以中和细菌生物膜。此外，铜绿假单胞菌产生的独特蛋白质的检测使细菌种类的鉴定，并可作为一种诊断方法在临床设置。鉴于耐药细菌菌株的出现和流行，快速诊断细菌感染促进快速和准确治疗的能力将减少耐药性的选择压力。此外，确定宿主对细菌入侵的免疫反应差异的能力增强了我们对眼表先天免疫系统调节的理解。在这里，我们描述了小鼠眼部感染和样本收集，以及概述了从角膜组织和细胞外环境(洗眼)样本中提取蛋白质和质谱分析的方案。©2020 Wiley期刊有限公司基本方案1:小鼠眼部感染模型基本方案2:小鼠模型样本采集基本方案3:从洗眼液中提取蛋白质基本方案4:从角膜组织中提取蛋白质基本方案5:从洗眼液和角膜组织样本中提取基于质谱的蛋白质组学和生物信息学

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Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.