The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by “top-down” CDMS measurements are 35–47% larger than those obtained from the “bottom-up” glycoproteomics studies, suggesting that the glycoproteomic measurements underestimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host’s immune system.

masses and relative abundances of impurities (i.e., empty and defective particles). CDMS is a robust technique that does not require sample-specific standards. The depth of information available from a CDMS assay that is quick (usually <1 h analysis time), efficient (sample size 10−20 μL), and sensitive enough to detect low abundance intermediates and misassembled particles has the potential to increase the speed and reduce the cost of vaccine development.

Heterogeneity usually restricts conventional mass spectrometry to molecular weights less than around a megadalton. As a single-particle technique, charge detection mass spectrometry (CDMS) overcomes this limitation. In CDMS, the mass-to-charge (m/z) ratio and charge are measured simultaneously for individual ions, giving a direct mass measurement for each ion. Recent applications include the analysis of viruses, virus-like particles, vaccines, heavily glycosylated proteins, and gene therapy vectors.

Recombinant adeno-associated virus (rAAV) is a leading gene therapy vector. However, neutralizing antibodies reduce its efficacy. Traditional methods used to investigate antibody binding provide limited information. Here, charge detection mass spectrometry (CD-MS) was used to investigate the binding of monoclonal antibody ADK8 to AAV serotype 8 (AAV8). CD-MS provides a label-free approach to antibody binding. Individual binding events can be monitored as each event is indicated by a shift of the antibody–antigen complex to a higher mass. Unlike other methods, the CD-MS approach reveals the distribution of antibodies bound on capsids, allowing AAV8 subpopulations with different affinities to be identified. The charge state generated by the electrospray of large ions is normally correlated with the structure, and the charge is expected to increase when an antibody binds to the capsid exterior. Surprisingly, binding of the first ADK8 to AAV8 causes a substantial decrease in the charge, suggesting that the first antibody binding event causes a significant structural change. The charge increases for subsequent binding events. Finally, high ADK8 concentrations cause agglutination, where ADK8 links AAV capsids to form dimers and higher order multimers.

The analysis of nucleic acids by conventional mass spectrometry is complicated by counter ions which cause mass heterogeneity and limit the size of the DNA that can be analyzed. In this work, we overcome this limitation using charge detection mass spectrometry to analyze megadalton-sized DNA. Using positive mode electrospray, we find two dramatically different charge distributions for DNA plasmids. A low charge population that charges like compact DNA origami and a much higher charge population, with charges that extend over a broad range. For the high-charge population, the deviation between the measured mass and mass expected from the DNA sequence is consistently around 1%. For the low-charge population, the deviation is larger and more variable. The high-charge population is attributed to the supercoiled plasmid in a random coil configuration, with the broad charge distribution resulting from the rich variety of geometries the random coil can adopt. High-resolution measurements show that the mass distribution shifts to slightly lower mass with increasing charge. The low-charge population is attributed to a condensed form of the plasmid. We suggest that the condensed form results from entropic trapping where the random coil must undergo a geometry change to squeeze through the Taylor cone and enter an electrospray droplet. For the larger plasmids, shearing (mechanical breakup) occurs during electrospray or in the electrospray interface. Shearing is reduced by lowering the salt concentration.