Accepted: OMASS Gault Robinson

Introduction

Cellular functions depend on coordinated activity of proteins with their binding partners. In their active state proteins typically exist in an ensemble of multiple conformations, which is modulated upon ligand binding. Identifying endogenous ligands that shift proteins into specific conformational states has a paramount importance in biology and is a key enabling factor in designing better therapeutics. Here we present a novel platform that uses selection in two dimensions, mass to charge (m/z) and ion mobility (IM) to simultaneously identity naturally bound ligands and corelate ligand binding with a specific conformational state. These results show the importance of ion mobility time alignment in deciphering the identity of multiple ligands and their coordinated activity in modulating protein conformational space.

Methods

A selection of proteins from different families (alpha helical and beta barrels) were selected to represent the vast diversity of protein structures. All proteins were expressed and purified in house, unless otherwise stated and protocols modified for enhanced preservation of ligand binding, including alterations in protein solubilisation time and temperature as well as the number of purification steps. All measurements were performed using a cyclic ion mobility enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. In-source activation conditions were optimised such that solvent or detergent micelles were removed but the bound ligands were preserved. Mobility aligned multistage MS was performed by combining IM separation with activation in pre- and post-IM sections of the instrument.

Preliminary Data

In order to first benchmark our IMS based platform for ligand identification, a known protein-ligand combination was used. The protein-ligand complex was activated to allow for sufficient desolvation in the source transfer optics prior to the quadrupole. The most intense protein-ligand ions were m/z selected and activated in the collision cell, resulting in a mixture of charge stripped protein-ligand complex, free protein, and singly charged ligand in the low m/z region. The released ligand was then mobility separated from the parent protein in the cyclic IM cell and further activated in the subsequent transfer collision cell generating ligand fragments. Since these fragments have the same arrival time as the detached ligand, their m/z values can be used to desipher the ligand identity.

We then extend this approach to more complex cohorts of ligands including different families of lipids and even those with different chain lengths. In these complex cases multiple passes around cyclic IM cell were sometimes necessary to sufficiently resolve detached ligands and to correlate fragments with their parent ligand.

To assess whether multiple conformational states of ligand bound proteins can be characterised using high resolution IM, a range of proteins were subjected to multi-pass separation. Interestingly, preliminary results showed no direct correlation between the mass, stoichiometry or protein family, with the final width of the arrival time distributions (ATD). We therefore suggest that the extent of the observed ATD broadening might report on the number of conformational states in which the protein can exist - an intrinsic property for each protein.

Further IM experiments are being performed to identify endogenous ligands that can be correlated to abundance of particular conformational states and the extent of ATD broadening. Our approach has the potential to reveal how natural ligands modulate protein dynamics.

Novel Aspect
A new IM based approach to simultaneously identify endogenous ligands and reveal how they can modulate protein conformations
Options:

A post-doc is presenting author on this abstract? Yes
A graduate student is presenting author on this abstract? No
An undergraduate student is presenting author on this abstract? No

Oral Choice:

Ion Mobility: Structure
Second Oral Choice:

Native MS in Structural Biology
Poster:

Ion Mobility: Applications

Submitting Author:

Joseph Gault
Oxford University
Oxford,
j.gault06@gmail.com