Construction of a High-Throughput Hydrogen Deuterium Exchange Mass Spectrometry Platform

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Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a structural biology technique which excels in the investigation of protein interactions. All proteins have amide groups along their polypeptide backbone which routinely undergo a phenomenon of solvent exchange - where the hydrogen of the amide swaps with the hydrogen of the surrounding solvent, typically water. The rate of this exchange is inextricably linked to the structural environment of that amino acid on the polypeptide backbone. Areas of protein which are rigid, densely packed and highly structured have a much slower solvent exchange rate than disordered, flexible and loosely packed parts of the protein. Additionally, the creating of new bonds through the creation of new interaction interfaces, whether that be protein: protein interactions, or protein: drug, protein: membrane, or protein: nucleic acid, will all alter the solvent exchange rate.The measurement solvent exchange is achieved through the exposure of a protein to a deuterated solvent. When a solvent exchange event occurs, the protein's hydrogen atom is swapped for deuterium, and thus the mass of the amino acid is altered. Using mass spectrometry equipment, we can detect this increase in mass, and by incubating the protein with deuterium for multiple time points, we can measure the solvent exchange rate. The solvent exchange rate can then be manipulated by e.g. adding a drug which interacts with the protein, and determine where on the protein the solvent exchange rate is altered, thus locate where on a protein a drug, binding partner, or lipid membrane is interacting. Determining how proteins interact with other entities can provide insights into their mechanisms, and can rationalise how diseases may alter their structural properties and impact their function. Likewise, determining where drugs interact with proteins can guide their development and inform how they may be altered to be more potent, or have fewer side-effects. HDX-MS has numerous advantages over other technologies which may also provide structural insights into how proteins interact with other agents. For example, the technique of x-ray crystallography is highly dependent on having a rigid protein structure, so proteins which are intrinsically disordered cannot be investigated. Another technology, cryo-electron microscopy, can overcome some of these limitations, but it is a very slow technique for screening molecules.This proposal is for a LEAP HDX Extended System automated sample handling robot, attached to a Waters Synapt XS HDMS Mass Spectrometer. Together these two pieces of equipment work seamlessly to produce the highest quality of HDX-MS data, allowing for the most complex and cutting-edge applications of the technique, while also ensuring the greatest efficiency of mass spectrometry capacity. The LEAP HDX Extended System allows for highly reproducible data collection and sample preparation. HDX-MS experiments are extremely sensitive to temperature fluctuations and incubation timing, making sample preparation ideally conducted by robotics. Additionally, the work-flow of chromatography involves multiple steps, again ideally conducted through automation to ensure reproducibility. Furthermore, the nature of HDX-MS experiments requires numerous samples being analysed individually on a single instrument, and a robotically handled sample injection system allows for a maximisation of useful data collection, especially through the night and over weekends. The Waters Synapt XS HDMS Mass Spectrometer is well suited for HDMS. Due to the temperature constraints on HDX samples, the ability to resolve peptides by chromatography is limited. The ion-mobility ability of the Synapt XS compensates for this when analysing complex mixtures of proteins. Additionally, the ability to use alternative fragmentation methods allows for more cutting edge HDX-MS experiments to be conducted which increase the resolution of the technique.