Combining nMS and native TDMS to investigate the involvement of native human mu opioid receptor in depression.

This project falls within the EPSRC physical sciences research area. G-protein coupled receptors (GPCRs), such as the mu opioid receptor (MOR), amount to close to 40% of drug targets.

GPCRs interact with ligands to elicit a response by selectively binding to a discrete G-protein, commencing a signalling cascade. Ketamine is a novel antidepressant, and it has been highlighted as a binding partner for MOR.

However, the mechanism determining the antidepressant response of ketamine binding to MOR is undetermined, with little prediction as to how the receptor selects a specific G protein upon ketamine binding.

Post-translational modifications (PTMs) add a further layer of complexity to the interactions of GPCRs, with their role in the selectivity of ligands and G protein binding yet to be fully elucidated.

In this project, I aim to (1) identify and characterise PTMs on native human MOR, and (2) explore the influence of such PTMs on interactions with ketamine and the G-proteins which bind to MOR (Gi family such as Gi1, Gi2 and Go).

Native mass spectrometry (nMS) is a powerful biophysical technique which enables intact protein complexes to be interrogated within the gas phase, allowing the varied stoichiometries of protein-protein and protein-ligand complexes to be determined, such as the binding of GPCRs to G-proteins to be observed.

With the study of membrane proteins such as MOR, these complexes often harbour a vast range of PTMs, such as glycosylation and lipidation, creating a plethora of individual proteoforms for a single protein complex. These individual proteoforms within the heterogenous protein populations can be hard to distinguish using nMS alone.

In these cases, native top-down mass spectrometry (nTDMS) can be used to sequence and characterise individual proteoforms.

Therefore, in this project, I will use combine the use of nMS and native TDMS to interrogate individual proteoforms and the role these play in protein and ligand interactions.

In order to investigate this, I will use a novel workflow similar to a recent study within the group into the proteoform specific interactions of bovine rhodopsin.

To meet Aim 1, I will first develop a method to express and purify native MOR in HEK293 cells which will produce receptor with PTMs such as glycosylation and potentially palmitoylation.

I will use a modified Tribrid Orbitrap mass spectrometer, customised to incorporate a 10.6 mu m laser directed into the linear ion trap (LIT).

By doing so, I can use IR photons to liberate MOR complexes directly from detergent micelles while retaining labile PTMs.

I will apply subsequent isolation and IR activation within the LIT, using IRMPD to fragment the receptors, providing sequence information, intact with PTMs, in order to identify and assign individual proteoforms.

Building from this in Aim 2, I will evaluate G protein binding to MOR in the presence of ketamine to deepen our understanding of the molecular mechanism by which ketamine may serve as an antidepressant.

In doing so, I introduce the possibility of identifying novel therapeutics for the treatment of depression to improve the treatment outlook for patients and further our understanding of mental health.