Mass spectrometry (MS) is a powerful technique for characterizing molecules, but analyzing macromolecular assemblies presents challenges due to inefficient transmission through mass analyzers and low resolving power at high mass-to-charge ratios (m/z). These large assemblies often exhibit heterogeneity due to variations in composition and post-translational modifications. Single-particle approaches, such as CDMS and nanoelectromechanical systems MS, offer advantages by circumventing the need to resolve complex ion signals. CDMS, in particular, provides an independent measure of the charge, overcoming charge state assignment bottlenecks. Recent work demonstrated Orbitrap-based CDMS, leveraging the linear relationship between ion charge, induced imaging current, and peak height in Fourier-transformed mass spectra. This opens opportunities to study single macromolecular ion behavior within the Orbitrap, expanding our current understanding primarily derived from ensemble measurements of smaller particles. Ion signal loss during mass analysis, due to unstable trajectories, collisions, or space charge effects, significantly impacts Orbitrap mass spectra sensitivity and quality. Collisions with background gas molecules are especially problematic for low m/z ions, leading to fragmentation and loss. However, high-mass ions exhibit surprising stability, even with long transient times. This study investigates the extraordinary stability of megadalton ions in the Orbitrap, exploring mechanisms of signal decay and developing improved data acquisition strategies for Orbitrap-based CDMS.

Native MS, using electrospray ionization under non-denaturing conditions, is crucial for analyzing macromolecular assemblies, preserving non-covalent interactions. It has been successfully applied to various systems, including ribosomal particles, membrane protein complexes, and viruses. While native MS provides valuable insights, the analysis of macromolecular assemblies is less efficient compared to smaller molecules. This is due to sub-optimal ion transmission and insufficient signal generation. Most mass analyzers also show reduced resolving power at higher m/z values, hindering the analysis of heterogeneous, high-mass samples. Single-particle approaches like CDMS and nanoelectromechanical systems MS have demonstrated impressive results for heterogeneous samples, but Orbitrap-based CDMS offers unique potential for investigating single macromolecular ion behavior.

The researchers adapted a 'frequency-chasing' method, previously used in FT-ICR mass spectrometry, to monitor individual megadalton ion behavior in the Orbitrap. This involved segmenting the recorded transients and performing separate Fourier transforms on each segment to track the frequency (and thus m/z) of individual ions over time. They analyzed single ions of HBV capsids (3-4 MDa), FHV (9.4 MDa), and a series of IgG1 oligomers (150-900 kDa) along with a 3 MDa nanocage. The instrument settings were optimized for high m/z ions, using various parameters to control ion transmission and desolvation. Transient recording times ranged from 128 ms to 4,096 ms. Data analysis included calculating mass resolution, signal-to-noise ratio, ion survival ratios, and quantifying neutral losses and charge-loss events. Modeling of the observed frequency shifts involved considering both linear and exponential decay functions to represent different stages of the activation process. Optimization strategies involved manipulating experimental parameters (pressure, activation, transient time) and implementing frequency drift corrections through the frequency chasing method to improve ion sampling and overall performance. Further analyses involved calculating ion path length, collisional cross-sections, and the average number of collision events.

Megadalton ions exhibited a surprising stability within the Orbitrap analyzer, surviving for seconds despite numerous collisions with background gas molecules. Mass resolution increased linearly with transient recording time, reaching >100,000 at 4,096 ms for HBV and FHV, enabling unprecedented mass precision. Frequency chasing revealed three distinct ion behavior types: stable ions, ions with gradual solvent loss, and ions undergoing a single charge-loss event. The gradual solvent loss was attributed to collisions with background gas molecules, while the charge-loss events allowed for direct mass and charge determination. Modeling of the frequency drifts showed a dual activation process: linear solvent loss due to collisions in the Orbitrap and exponential loss during ion injection from the C-trap. Optimization strategies, including pressure reduction, improved desolvation, shorter transients, and frequency chasing, led to a significant increase (~23-fold) in effective ion sampling and a twofold improvement in resolution. Finally, the analysis of long transients revealed radial ion motion through frequency modulation of the axial frequency, confirming theoretical predictions.

The unexpected stability of megadalton ions in the Orbitrap, contrasting with the behavior of smaller ions, results from the attenuation of collision energy by gradual solvent loss rather than fragmentation. This finding highlights the differences in energy dissipation mechanisms between large and small ions. The detailed understanding of ion activation processes, combined with frequency chasing, enabled substantial improvements in Orbitrap-based CDMS performance. The improved mass resolution and precision have significant implications for various research areas, particularly structural biology and virology, where the analysis of heterogeneous macromolecular assemblies is critical. The use of frequency-chasing provides a path to extend the transient time and further improve performance, although hardware limitations currently restrict the maximum to 4 seconds. Future development in this technology could lead to nearly perfect charge assignment.

This study reveals the remarkable stability of megadalton ions in the Orbitrap, leading to significant advancements in Orbitrap-based CDMS. The frequency-chasing method effectively mitigates the effects of frequency drift, dramatically improving ion sampling and mass resolution. Future work should focus on overcoming current hardware limitations to extend transient recording times and explore further performance improvements. This will enhance the capabilities of CDMS for studying complex macromolecular systems.

The current study is limited by the maximum transient time achievable on the available instrument (4 seconds). Further optimization of experimental parameters and the development of new hardware could potentially push the limits of resolution and precision even further. The analysis focused on a specific set of macromolecular systems; the generalizability of the findings to other systems should be further investigated. The frequency chasing method, while effective, requires considerable computational processing and might be time-consuming for very large datasets.