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Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry

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Journal Article - Open Access

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Utrecht University Utrecht Netherlands

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Since the concept was first introduced by Brian Chait and co-workers in 1991,mass spectrometry of proteins and protein complexes under non-denaturing conditionsnative MS has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton MDa protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses the Brome Mosaic Virus BMV and the Cowpea Chlorotic Mottle Virus CCMV. Both CCMV and BMV are approximately 4.6 megadalton MDa in mass, of which approximately 1MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass2 . This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible.

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  • Microbiology

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