The mass spectrometry facility runs routine HDX analyses to determine protein folding states, effects of ligand binding and protein interactions (e.g. epitope mapping). It also has a covalent labelling setup for hydroxyl radical foot printing via Fast Photochemical Oxidation of Proteins (FPOP) linked to mass spectrometry, to probe changes in solvent accessibility. Further advanced instrumentation offers bespoke high-resolution native MS and ion mobility capabilities: Ultra-high mass range Q-Exactive Plus Orbitrap enabling native analysis of heterogeneous systems at high mass-to-charge ratios, e.g. membrane protein lipid complexes, and Tofwerk high-resolution ion mobility TOF (in late 2018) as well as several Waters Synapt HDMS systems.
Incubation of protein samples in deuterated water with subsequent MS measurement can elucidate information pertaining to an analyte protein's higher-order structure. The rate of exchange of amide back-bone protons with deuterons in the bulk solvent is a function of solvent accessibility and hydrogen bonding when temperature and pH are carefully controlled. These rates are affected by secondary, tertiary and quaternary structure and can be used to locate changes in protein conformation and dynamics as well as of binding interfaces between interacting partners.
Using Waters M-Class UPLC and Synapt G2S-i mass spectrometer purchased with funds from the BBSRC, we have developed robust protocols for performing comparative HDX-MS at the peptide level to probe a range of biologically relevant systems including protein-ligand interactions, nanobody & affimer binding and membrane proteins.
The Biomolecular Mass Spectrometry Facility provides access to the hydrogen/deuterium exchange (HDX)-MS capabilities. The instrumentation consists of LEAP sample handling robot, Waters UPLC with HDX Manager and Waters Synapt G2-Si. Data processing uses ProteinLynx Global Server and DynamX.
We perform peptide level analysis using online pepsin and aspergillopepsin proteolysis. Labelling durations can be chosen between 30s and 8h however in most cases 60 min should suffice as the maximum incubation time. Protein concentration is optimised during preliminary experiments. A minimum protein concentration of 10 µM and volume 150 uM being is required however higher stock concentrations are encouraged. Runs for each labelling condition takes approx. 16-18 hours depending on incubations chosen and the number of replicates. An additional day of access should be requested for instrument setup and preliminary experiments.