Scientific Core 2:Mass Spectrometry and Biophysics

点击翻译按钮获取中文摘要

Scientific Core 2 (SC2). Project Summary/Abstract The application of mass spectrometry and biophysical approaches to various facets of this Program Project is a unique aspect of this research program. The purpose of Scientific Core 2 (SC2) is to provide both mass spectrometric and biophysical support for the investigators within the Program Project. A principal objective of SC2 is to make available robust, state-of-the-art techniques for accurate and reproducible measurement of macromolecular interactions and protein physical properties. Addressing this objective, the Core serves the common need of all three Projects for investigation of protein characteristics, conformational changes, and protein-protein interactions. A second principal objective is mass spectrometric detection and quantification of small molecules, including bioactive, oxidized lipids and oxidized amino acids that are reporters of oxidative processes. SC2 serves as a centralized area of expertise in analytical, structural, and modeling/computational biophysics. The Core provides not only access to instrumentation and reagents, but more importantly provides experience and expertise in setting up the methods, analyzing and interpreting the results, and assuring quality control. The Core consists of two major sub-components with different but highly complementary methods and approaches: a Mass Spectrometry Sub-component and a Biophysics Sub-component. SC2 will provide expertise and reagents for detection of direct interaction by surface plasmon resonance spectrometry that reports accurate determinations of on- and off-rates of binding, as well as dissociation constants, KD. This method is complemented by isothermal titration calorimetry for thermodynamic characterization of macromolecular assemblies. The Mass Spectrometry Sub-component of SC2 will facilitate identification of protein-protein interaction sites by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), a powerful tool that also provides information on protein dynamics. This approach is supplemented by two methods provided by the Biophysics Sub-component - Förster resonance energy transfer and the tethered artificial protease procedure (“Fe-BABE”). The Core provides expertise in modeling of proteins and their interactions using known structures, protein-protein docking methods, and molecular dynamics simulations. Mass spectrometry provides a powerful discovery tool in proteomic analysis useful in identification of proteins as well as post-translational modifications. Alternative mass spectrometry instrumentation will be used for detection and quantification of small molecules, including bioactive oxidized lipids and modified amino acids that are specific reporters of oxidative stress. SC2 will provide a diversity of services including provision of reagents, assistance in probe design and construction, design and performance of experiments, and data analysis and interpretation of results. An educational component is an additional strength of this Core. All methods provided by SC2 require highly specialized instrumentation, technical expertise, and experience not generally available in a modern cell or molecular biology laboratory; additionally, some methods require unfamiliar computational analysis methods. Thus, the Mass Spectrometry and Biophysics Core provides critical capabilities essential for the success of the Program Project.