Mass Determination of Filled and Empty AAV5 Particles Enabled by Nanoelectrospray Ionization and Proton Transfer Charge Reduction

The mass determination of bio-ions that exceed a megadalton poses many challenges. While it is possible to generate gaseous ions from large biocomplexes, including intact viruses, via nanoelectrospray ionization (nESI), generating mass information using conventional ensemble measurements (i.e., from conventional mass spectra) requires the resolution of charge states. As biocomplexes increase in size, overlap of adjacent charge states becomes increasingly problematic. Single ion measurements that enable the simultaneous determination of mass/charge and charge can overcome the charge state overlap problem. However, ensemble measurements are, in principle, much faster. We demonstrate here the mass determination of empty and filled adeno-associated virus particles, serotype 5 (AAV5), both separately and as a mixture using nESI, gas-phase proton transfer ion/ion reactions, and time-of-flight mass analysis. The ion/ion reactions are used to reduce charge states to the point at which they can be resolved, and UniDec, a publicly available deconvolution program, is used to facilitate mass determination. This work demonstrates that mass measurements of binary mixtures of empty and filled AAV5 particles as large as 3.7–4.5 MDa can be enabled via the use of single proton transfer ion/ion reactions to facilitate charge state resolution.