Project description:Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time consuming. Moreover, the HDX community is expanding to investigate multi-protein and highly complex protein systems such as cell lysates which further complicates data analysis. Thus, a simple and easy-to-use open source software package designed to analyze large and highly complex protein systems is needed. In this vein, we have developed The Deuterium Calculator, a Python-based software package for HDX-MS data analysis. The Deuterium Calculator is capable of differential and nondifferential HDX-MS analysis, produces standardized data files according to the recommendations put forth by the International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX) to increase transparency in data analysis, and generates Woods’ plots for statistical analysis and data visualization. This standard output can be used to perform further analysis on HDX such as determination of labeling time dependent deuteration and for the study of protein folding kinetics or differential uptake. Moreover, the Deuterium Calculator is capable of analyzing large HDX-MS datasets (e.g., LC-HDX-MS from complex samples such as cell lysates) to determine the extent of deuteration on individual peptides from numerous proteins, perform differential analysis under varying experimental conditions, and time-dependent deuterium exchange. The Deuterium Calculator is freely available for download at https://github.com/OUWuLab/TheDeuteriumCalculator.git.
Project description:We employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the conformational dynamics required to facilitate based Brownian ratchet mechanism for protein secretion (by the SecA-SecYEG complex).
Project description:Hydrogen deuterium exchange mass spectrometry of HSL in the presence of artificial lipid droplets to analyze lipid droplets binding.
Project description:Hydrogen deuterium exchange mass spectrometry of PLIN3 in the presence of three different membrane vesicles to analyze structural changes induced by membrane binding.
Project description:Hydrogen/deuterium exchange mass spectrometric methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in the deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues.