Core B: Mucus/Mucin Analytics Core

点击翻译按钮获取中文摘要

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.