Surface-induced dissociation of protein complexes on a cyclic ion mobility spectrometer.

Dalton T Snyder,Benjamin J Jones,Darren Hewitt,Jason Wildgoose,Dale A Cooper-Shepherd,Vicki H Wysocki,Yu-Fu Lin,Jeffery M Brown,James I Langridge

doi:10.1039/d1an01407b

Dalton T Snyder, Benjamin J Jones + Show 7 more

Open Access

https://doi.org/10.1039/d1an01407b

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Abstract

We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20–50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after ∼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared ‘native’ in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.

Highlights

We find an overall increase in mobility resolution compared to the Synapt G2 and show that increasing the pathlength further via multipass separations around the cyclic ion mobility spectrometer (cIMS) array is useful for resolving heterogeneous populations of Collision-induced unfolding (CIU) and surface-induced unfolding (SIU) conformers
While multipass separations are clearly beneficial for CIU and SIU experiments of protein assemblies, for Surface-induced dissociation (SID) we found that another very useful feature of the cIMS was the improved single pass mobility resolving power for deconvoluting fragments that overlap in m/z, which are abundant in SID spectra of cyclic complexes due to symmetric charge partitioning.[54,55]
The SID cell is wholly integrated into the hardware and software on the cIMS platform, requiring only a single input (SID collision voltage) from the user, an attractive improvement over more complicated SID cells that we have previously developed for native mass spectrometry

Summary

Introduction

Native mass spectrometry (nMS) is a growing field that provides information complementary to that acquired with traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance.[1,2] nMS is a powerful technique that allows the study of proteins and protein complexes in the gas phase by kinetic trapping of solution structures during the electrospray process,[3] enabling the determination of stoichiometry, ligand binding, oligomeric state, and, when coupled with ion mobility, collision cross section measurements (which can be compared to structures determined by other structural biology tools or computational structure predictions).[4,5,6]Coupling native mass spectrometry with activation methods such as collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) allows structure and sequence information to be determined.[7,8,9,10] Surface-induced dissociation (SID) allows ligand binding and subunit connectivity information to be obtained as well.[11,12,13] SID typically produces substructure fragments that are reflective of the native solution structure of the protein complex of interest.

Results

Conclusion