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

Michiel Van De Waterbeemd,Albert J R Heck,Bogdan G Dragnea,Irina B Tsvetkova,Jeroen J Cornelissen,Joost Snijder

doi:10.1007/s13361-016-1348-6

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 ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1348-6) contains supplementary material, which is available to authorized users.

Highlights

Mass spectrometry of protein complexes under non-denaturing, near-native conditions has advanced strongly since the concept was first reported by Brian Chait and co-workers in 1991 [1]
Chlorotic Mottle Virus (CCMV) and Brome Mosaic Virus (BMV) are multipartite viruses, which mean that the end result of infection is a mixture of particles that consist of the same protein capsid encapsulating different segments of the genome (Figure 1e)
We test whether and how we are able to dissect the mass heterogeneity in BMV and CCMV by high resolution native mass analysis making use of both quadrupole time-offlight (Q-TOF) and Orbitrap mass analyzers and organic additives to manipulate the charging of these assemblies during the electrospray process

Summary

Introduction

Mass spectrometry of protein complexes under non-denaturing, near-native conditions (native MS) has advanced strongly since the concept was first reported by Brian Chait and co-workers in 1991 [1]. 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.

Results

Conclusion