Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

Charles Eldrid,Rehana Akter,Aisha Ben-Younis,Kevin Giles,Hannah Britt,Daniel Raleigh,Nick Tomczyk,Mike Morris,Symeon Kalfas,Dale Cooper-Shepherd,Jakub Ujma,Konstantinos Thalassinos,Tristan Cragnolini

doi:10.1021/jasms.1c00018

Abstract

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IMn) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IMn-MS experiments also show evidence of interconversion between compact and extended structures. IMn-MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.

Highlights

IntroductionBackground data collection was performed by keeping the initial slice-collision activation (CA) injection sequence parameters used for data collection and setting the “eject to prearray store” function to 0.01 ms so that a negligible quantity of ions are stored
Presents as a single feature with an extended tail. This population of ions has been previously subjected to multiple passes around the cIM; we were not able to resolve them into more distinct features.[27]
The experiments enabled by the Q-cIM-ToF instrument described here reveal insights into the conformational behavior of proteins in the gas phase and overcome the major limitation highlighted in our previous work, which is that increased resolution for arrival time distribution (ATD) produced from protein ions is limited due to the width and complexity of the conformers present.[27]

Summary

Introduction

Background data collection was performed by keeping the initial slice-CA injection sequence parameters used for data collection and setting the “eject to prearray store” function to 0.01 ms so that a negligible quantity of ions are stored. To keep the injection sequence times the same, the “eject” function was increased to compensate for the shortened storing function (Figure S1). Each voltage increment of each slice and background was collected for 250 scans exactly. Before each sequence of events occurring in the cIM, ions are accumulated in the trap ion guide (Figure 1) to ensure high duty cycle. To verify that conformational changes were not occurring during accumulation in the trap, ions were held there for varying times before a single pass separation, as described previously.[27]

Methods

Results

Conclusion