Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies

Abstract

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (<i>K</i>₀ = 0.185-1.84 cm²·V^-1·s^-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (<i>Sr</i>) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCS_N₂) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (<i>n</i> = 6-73), Tuning Mix oligomers (<i>n</i> = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (<i>n</i> = 1-4), carbonic anhydrase, β clamp (<i>n</i> = 1-4), topoisomerase IB, bovine serum albumin (<i>n</i> = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (<i>n</i> = 1-2)) covering a wide mass (up to <i>m</i>/<i>z</i> 19 000) and CCS range (up to 22 000 Ų with <0.6% relative standard deviation (RSD)).