Eatough, D. | Search bioRxiv

bioRxiv2026

A Charge Detection Mass Spectrometer for the Analysis of Megadalton-sized Molecules

Advances in Electrostatic Linear Ion Trap (ELIT) Charge Detection Mass Spectrometry (CDMS) over the past 10 years have revolutionized its use for analyzing very high-molecular-weight species such as protein complexes, viral vectors, vaccines, viruses, and amyloid fibrils. Nonetheless, ELIT-based CDMS has remained confined to a small number of specialized instrumentation groups, predominantly in academia, where large and complex home-built instruments are operated by highly skilled scientists in dedicated facilities. In this report, we discuss the primary challenges addressed in the design of a benchtop ELIT-based CDMS instrument. We highlight key design aspects of the hardware, acquisition modes, and control software, and we present important performance metrics (mass range, resolution, and sensitivity) demonstrated using samples representative of the technologys key application areas.

biophysics↗

bioRxiv2026

Applying distinct CDMS strategies to observe non-classical virus capsid assembly

In conventional native mass spectrometry (MS), one faces severe limitations when challenged with heterogenous, high mass samples, commonly failing to resolve clear peak distributions and thus mass determination. Charge detection MS (CDMS) has emerged as a premier method to analyze these samples by determining mass-to-charge ratio (m/z) and charge (z) simultaneously. Here, the two currently available commercialized CDMS systems, the Orbitrap-based Direct Mass Technology (DMT) and the electrostatic linear ion trap (ELIT)-based Xevo CDMS are applied to human norovirus capsids from two different strains, GI.1 Norwalk and GII.17 Kawasaki. The norovirus capsid is highly heterogenous due to N-terminal processing on the repeating subunits that it is built from and commonly forms T = 3 and sometimes T = 4 particles. Both CDMS approaches were able to determine similar masses in both strains. GII.17 Kawasaki exhibits both T = 3 and T = 4 particles, though the Xevo CDMS measurements were closer to the theoretical mass than the DMT instrument. Interestingly, GII.17 Kawasaki also displayed non-classical mass distributions with high abundance in-between T = 3 and T = 4 which was then confirmed by cryogenic electron microscopy (cryo-EM), demonstrating an oval capsid shape. GI.1 Norwalk displays a wide mass distribution in both instruments that exceeds the theoretical T = 3 mass by 8-10 %. Proteomics and native MS experiments suggest possible interactions with a protein from the expression system. This study demonstrates the capabilities of two distinct CDMS methodologies on two viral capsids and presents the first non-classical capsid assembly in a GII.17 noroviral capsid.

biochemistry↗