title: Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry.
abstract: Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native 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 1 MDA 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 mass (~2%). 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. Graphical Abstract ᅟ.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH, Utrecht, The Netherlands.|Netherlands Proteomics Center, Padualaan 8, Utrecht, 3584 CH, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: van de Waterbeemd
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH, Utrecht, The Netherlands.|Netherlands Proteomics Center, Padualaan 8, Utrecht, 3584 CH, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Snijder
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA.
authors.forename: Irina B
authors.initials: IB
authors.lastname: Tsvetkova
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA.
authors.forename: Bogdan G
authors.initials: BG
authors.lastname: Dragnea
authors.affiliation: Laboratory for Biomolecular Nanotechnology, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE, Enschede, The Netherlands.
authors.forename: Jeroen J
authors.initials: JJ
authors.lastname: Cornelissen
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH, Utrecht, The Netherlands. a.j.r.heck@uu.nl.|Netherlands Proteomics Center, Padualaan 8, Utrecht, 3584 CH, The Netherlands. a.j.r.heck@uu.nl.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D017795:Bromovirus / Q000235:genetics*; D013058:Mass Spectrometry; D012367:RNA, Viral / Q000032:analysis*
chemical_list: D012367:RNA, Viral
keywords: Brome Mosaic Virus (BMV); Cowpea Chlorotic Mottle Virus (CCMV); Genome; Native mass spectrometry; Orbitrap; Q-TOF; Virus