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

Abstract

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.

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology

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“…Including the structural proteins (816 copies), the cement proteins (∼785 copies), and the core proteins (∼998 copies), there are expected to be ∼2 599 proteins in HAdV5. From the sequences and copy numbers, the expected mass of HAdV5 is 142.5 MDa, including the dsDNA genome …”

“…From the sequences and copy numbers, the expected mass of HAdV5 is 142.5 MDa, including the dsDNA genome. 235 CDMS measurements of HAdV5 show a sharp peak at 156.1 MDa that is attributed to the mature virus and a smaller peak at 129.6 MDa (see Figure 11a). 235 The 129.6 MDa peak is due to empty or light particles that do not contain the genome but may contain packaging and proteins that are not present in the mature virion.…”

“…235 CDMS measurements of HAdV5 show a sharp peak at 156.1 MDa that is attributed to the mature virus and a smaller peak at 129.6 MDa (see Figure 11a). 235 The 129.6 MDa peak is due to empty or light particles that do not contain the genome but may contain packaging and proteins that are not present in the mature virion. 236,237 Light and heavy particles are separable by gradient centrifugation.…”

Effects of Molecular Size on Resolution in Charge Detection Mass Spectrometry

“…However, for much larger protein complexes and viruses weighing 500+ kDa, the resolution achieved by conventional MS methods begins to be limited by the intrinsic heterogeneity of the sample. ,, Extrinsic factors, such as the extent of adduction of salts and/or solvent left over from the ionization process, have been shown to often add ∼1% or more to the overall mass of ions in this size range, further complicating accurate mass determinations . As the molecular size extends into the MDa regime and intrinsic heterogeneity increases, conventional MS techniques begin to fail due to m / z peak congestion, making individual ion techniques, such as CDMS, the only option for rapid mass measurements. − CDMS has been used to analyze the masses of often complex mixtures of analytes with masses ranging from a few thousand Da to 500+ MDa. ,,− …”