Extending the boundaries of native mass spectrometry to study virus structure and assembly

Abstract

Native mass spectrometry (MS) is a powerful tool to study the composition and quaternary structure of protein complexes over a wide range of size and mass. As an analytical tool, native MS offers unmatched specificity and precision to pinpoint the stoichiometry of biomolecular complexes. It has been developed for analysis of larger protein complexes, such as intact virus particles, in recent years. The use of native MS was shown to be particularly convenient to characterize the assembly and composition of these large particles, offering important information on their role as pathogens and their use for nanotechnology and medicine. This thesis describes the development and application of native MS to analyze the structure and assembly of virus particles. It is shown that virus particles as big as 18 megadalton can be analyzed with native MS on a modified quadrupole time-of-flight instrument. Analysis of the obtained peak shapes and theoretical considerations about the inherent spread in masses and the performance of the mass analyzer lead to the conclusion that the 18 megadalton particles are at the limit of what is feasible on the current generation of instruments, and shows that poor desolvation of the ions is currently the main limiting factor. Having established that 18 megadalton capsids can be analyzed by native MS, several maturation intermediates of bacteriophage HK97 were analyzed by native MS to learn about the maturation pathway of the virus. It is furthermore shown how we extended the mass range of a novel type of Orbitrap mass analyzer for native MS to allow analysis of virus particles of several megadaltons. The transmission at high mass-to-charge ratio is improved by modifying the ion guides of the instrument. Sensitivity is improved 5-fold in the high m/z range to allow analysis of virus particles with improved resolution compared to the time-of-flight based platforms. It is demonstrated that the Orbitrap with extended mass range can be used to quantify cargo loading in a virus-like particle for nanotechnology and that it can be used to determine the stoichiometry of capsids in heterogeneous mixtures. The use of both the quadrupole time-of-flight and the Orbitrap-based instrument is demonstrated in this thesis in three applications to virus structure and assembly: native mass spectrometry is used in combination with proteomics-type LC-MS/MS, hydrogen-deuterium exchange MS and atomic force microscopy to uncover a novel component of mature adenovirus, to study assembly and uncoating of triatoma virus and to study the effects of cargo encapsulation in a virus-like bacterial nanocompartment called encapsulin. This thesis thereby illustrates the great potential of native MS to study virus capsid structure and assembly. Several potential improvements of current native MS instrumentation as well as possible fundamental limitations of the technique are discussed to establish the current and future role of native MS in the analytical toolbox to study protein complex structure and assembly.