Applying Distinct CDMS Strategies to Observe Nonclassical Virus Capsid Assembly

In conventional native mass spectrometry (MS), one faces severe limitations when challenged with heterogeneous, 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 heterogeneous 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 nonclassical 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 nonclassical capsid assembly in a GII.17 noroviral capsid.