Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa.

Adenovirus is one of the largest nonenveloped, double-stranded DNA viruses. It is widely used as a gene therapy vector and has recently received a lot of attention as a novel vaccine platform for SARS-CoV-2. Human adenovirus 5 (HAdV5) contains over 2500 protein molecules and has a 36 kbp genome. Adenovirus is well beyond the range of conventional mass spectrometry, and it was unclear how well such a large complex could be desolvated. Here, we report molecular weight (MW) distributions measured for HAdV5 and for 11 recombinant AdV vectors with genomes of varying lengths. The MW distributions were recorded using ion trap charge detection mass spectrometry (CDMS), a single-particle technique where m/z and charge are measured for individual ions. The results show that ions as large as 150 MDa can be effectively desolvated and accurate MW distributions obtained. The MW distribution for HAdV5 contains a narrow peak at 156.1 MDa, assigned to the infectious virus. A smaller peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome. The ions in the 129.6 MDa peak have a much lower average charge than those in the peak at 156.1 MDa. This is attributed to the empty particles missing some or all of the fibers that decorate the surface of the virion. The MW measured for the mature virus (156.1 MDa) is much larger than that predicted from sequence masses and copy numbers of the constituents (142.5 MDa). Measurements performed for recombinant AdV as a function of genome length show that for every 1 MDa increase in the genome MW, the MW of the mature virus increases by around 2.3 MDa. The additional 1.3 MDa is attributed to core proteins that are copackaged with the DNA. This observation suggests that the discrepancy between the measured and expected MWs for mature HAdV5 is due to an underestimate in the copy numbers of the core proteins.

Heterogeneity of Glycan Processing on Trimeric SARS-CoV-2 Spike Protein Revealed by Charge Detection Mass Spectrometry.

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.