Accepted: Waters internal protein unfolding

Introduction

Collision-induced unfolding (CIU) is a technique that has gained interest in the fields of life science and biopharmaceutical research. The technique involves subjecting gas phase protein ions to elevated activation energies in order to induce structural changes that are monitored by ion mobility-mass spectrometry. Published work has shown that the resulting unfolding profiles relate closely to protein domain architecture1, and studies on biopharmaceuticals have shown differences between different immunoglobulin classes2. Here, we present work that employs a cyclic ion mobility (cIM)-enabled quadrupole time-of-flight mass spectrometer to perform novel tandem ion mobility (tandem-IMS) experiments, probing protein unfolding pathways in greater detail.
1. Zhong et al, Angewandte. 2014, 12, 35, 9363-9366
2. Tian et al, Anal. Chem. 2015,87, 22, 11509-11515

Methods

Studies were performed on a cyclic ion mobility-enabled Q-ToF (Q-cIM-oaToF) mass spectrometer. The cIM device has multiple benefits: the circular path provides a longer, higher mobility resolution separation path; a multi-pass capability provides significantly higher resolution, albeit over a reduced (selected) mobility range, the multifunctional ion entry/exit array can selectively eject species within a range of mobilities, providing additional tandem-IMS functionality. Tandem-IMS enables both mass and mobility precursor selection, with mobility and mass measurement of the resulting activated species, providing improved selectivity in CIU studies. Human transthyretin (TTR) was used as a model system in this study. The protein was electrosprayed from conductive-coated glass capillaries at a concentration of 4 micromolar in 200 mM ammonium acetate.

Preliminary Data

Protein ions under study were mass-selected in the resolving quadrupole to allow the selective unfolding of a single charge state at a time. Initially focussing on the 16+ charge state, six passes of the cIM device were performed (approximately 6m pathlength) with low activation voltage. No additional features were observed in the arrival time distribution in comparison to a single pass, consistent with there only being one family of conformations populated by this charge state. This is in agreement with the general observation that protein ions form an ensemble of closely related conformations that do not necessarily separate under high mobility resolution conditions.

Protein unfolding was induced by increasing the acceleration voltage into the trap ion guide situated before the cIM device. At low voltages a single species was observed in the arrival time distribution. Increasing the voltage to 40 V resulted in a minimum of four species (1-4 from most compact to most extended) being observed in the arrival time distribution after a single pass of the cIM device. The multifunctional array was used to isolate and eject each of the four species to the pre-array store (mobility-based selection). The protein ions were subsequently reinjected into the cIM device at elevated energies to induce further unfolding. By mobility-selecting each of the four species after a single cIM pass and activating on reinjection, the initial unfolding pathway was reproduced. Briefly, after selecting species 1, species 2-4 were formed; on selecting species 2, species 3 and 4 were formed; upon selecting species 3, species 4 was formed. Interestingly, no compacted intermediates were observed for TTR. Other model systems will be discussed.

Novel Aspect
Tandem ion mobility-mass spectrometry for detailed protein unfolding studies

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Poster:

Proteins: Complexes/Non-covalent Interactions

Submitting Author:

Dale Cooper-Shepherd
Waters Corporation
Wilmslow,
dale_cooper-shepherd@waters.com