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Resolving heterogeneous high-mass macromolecular machineries by Orbitrap-based single particle charge detection mass spectrometry

By Tobias P. W├Ârner, Joost Snijder, Antonette Bennett, Mavis Agbandje-McKenna, Alexander A Makarov, Albert J.R. Heck

Posted 28 Jul 2019
bioRxiv DOI: 10.1101/717413

Here we show that single particle charge-detection mass spectrometry (CD-MS) can be performed on a ubiquitous Orbitrap mass analyser and applied to the analysis of high-mass (megadalton) heterogeneous biomolecular assemblies. We demonstrate that single particle high-mass ions can survive in the Orbitrap for seconds, whereby their measured signal amplitudes scale linearly with charge state over the entire m/z range. Orbitrap based single particle CD-MS can be used to resolve mixed ion populations, accurately predict charge states, and consequently also the mass of the ions. We successfully applied CD-MS to challenging natural and biotherapeutic protein assemblies, such as IgM oligomers, designed protein nano-cages, ribosome particles and intact, empty- and genome-loaded Adeno-associated virus particles. Single particle CD-MS combined with native MS on existing Orbitrap platforms will greatly expand its application, especially in the mass analysis of megadalton heterogeneous biomolecular assemblies.

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