Core B: Mucus/Mucin Analytics Core

核心 B：粘液/粘蛋白分析核心

• 批准号: 10684187
• 负责人: Mehmet Kesimer
• 金额: $ 26.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684187
• 关键词: Adhesions; Agar Gel Electrophoresis; Biochemical; Biophysics; Cell Culture Techniques; Charge; Cilia; Coupled; Disease; Doctor of Philosophy; Dryness; Electron Microscopy; Epithelium; Gel; Gland; Goblet Cells; Health; Human; Human Resources; Investigation; Label; Laboratories; Lasers; Left; Liquid substance; MUC5AC gene; MUC5B gene; Mass Spectrum Analysis; Measurement; Measures; Medicine; Modeling; Molecular; Molecular Sieve Chromatography; Molecular Weight; Mucin 1 protein; Mucin-2 Staining Method; Mucins; Mucous body substance; Mus; North Carolina; Pathology; Performance; Phenotype; Positioning Attribute; Property; Proteins; Proteome; Proteomics; Refractometry; Research Personnel; Resources; Respiratory Mucosa; Sampling; Services; Shapes; Solid; Specific qualifier value; Structure; Submucosa; Surface; Techniques; Training; Ultracentrifugation; Universities; Weight; Western Blotting; air sampling; dimer; in vivo; instrumentation; light scattering; molecular size; professor; respiratory; success; viscoelasticity; zeta potential

Abstract The Mucus/Mucin Analytics Core will perform key mucin/mucus biochemical, biophysical, and proteomics measurements for all the Projects of the PPG. These services are required for our PPG investigators who are focused on a mechanistic understanding of the physical and biochemical forces which govern mucus viscoelasticity, transport, and the adhesion of mucus to epithelial surfaces. Qualitative and quantitative characterization of mucus secretions from culture models, submucosal gland mucus, and Muc5b dimer and WT mice and their MTEC cultures are key for successful execution the projects and for the overall synergistic success of this PPG. Mucus and mucin measures raise unique challenges in their performance from biochemical, biophysical, and proteomic perspectives. Therefore, the mucus/mucin analytics core is a centralized resource for aiding all the PPG projects with their mucin and mucus analysis requirements. The Mucus/Mucin analytics Core will offer the following services to the Projects: 1- Mucin/Mucus quantitation: Total mucin concentrations, total biomolecule concentrations and solids (%) dry weight measurements of overall mucus concentration in mucus samples will be measured. Absolute concentration of mucins MUC5AC and MUC5B, and other airways innate defense proteins, including PRR4, LPLUNC1, FCGBP, will be measured by labelled mass spectrometry. 2- Mucin characterization: Isolation of mucin molecules and characterization of their macromolecular properties, including molecular weight (MW), hydrodynamic size (Rg), charge status, are key parameters for the necessary calculations made by the projects. Molecular weight and size, oligomerization status, shape (compact vs linear), charge status will be assessed by GPC, refractometry, multi-angle laser light scattering (MALLS), western blotting following agarose gel electrophoresis, and rate zonal ultracentrifugation followed by electron microscopy. 3- Mucin/Mucus proteome: Mass spectrometry-based proteomics characterization of culture secretions and, gland secretions, and the proteins comprising the mucin-interacting proteome will be identified.

摘要 粘液/粘蛋白分析核心将执行关键的粘蛋白/粘液生化，生物物理和蛋白质组学 PPG所有项目的测量。我们的PPG研究者需要这些服务， 集中于对控制粘液的物理和生物化学力量的机械理解 粘弹性、运输和粘液对上皮表面的粘附。定性和定量 来自培养模型的粘液分泌物、粘膜下腺体粘液、Muc 5 b二聚体和WT的表征 小鼠及其MTEC培养物是成功执行项目和整体协同的关键。 这是PPG的成功。粘液和粘蛋白测量在其性能方面提出了独特的挑战， 生物物理学和蛋白质组学观点。因此，粘液/粘蛋白分析核心是集中的资源， 帮助所有PPG项目满足其粘蛋白和粘液分析要求。粘液/粘蛋白分析 核心将提供以下服务的项目：1-粘蛋白/粘液定量：总粘蛋白浓度， 总生物分子浓度和固体（%）总粘液浓度的干重测量 将测量粘液样品。粘蛋白MUC 5AC和MUC 5 B以及其他气道的绝对浓度 将通过标记质谱法测量先天防御蛋白，包括PRR 4、LPLUNC 1、FCGBP。 2-粘蛋白表征：分离粘蛋白分子并表征其大分子性质， 包括分子量（MW）、流体动力学尺寸（Rg）、电荷状态，是必要的 项目进行的计算。分子量和大小，低聚状态，形状（紧凑型与线性）， 电荷状态将通过GPC、折射测定法、多角度激光散射（MALLS）、Western印迹法和免疫组织化学法进行评估。 琼脂糖凝胶电泳后进行印迹，并进行速率区带超离心，然后进行电子显微镜检查。 3-粘蛋白/粘液蛋白质组：培养分泌物的基于质谱的蛋白质组学表征， 腺体分泌物以及构成粘蛋白相互作用蛋白质组的蛋白质将被鉴定。