Characterizing Heterogeneous Mixtures of Assembled States of the Tobacco Mosaic Virus Using Charge Detection Mass Spectrometry

Abstract

The tobacco mosaic viral capsid protein (TMV) is a frequent target for derivatization for myriad applications, including drug delivery, biosensing, and light harvesting. However, solutions of the stacked disk assembly state of TMV are difficult to characterize quantitatively due to their large size and multiple assembled states. Charge detection mass spectrometry (CDMS) addresses the need to characterize heterogeneous populations of large protein complexes in solution quickly and accurately. Using CDMS, previously unobserved assembly states of TMV, including 16-monomer disks and odd-numbered disk stacks, have been characterized. We additionally employed a peptide−protein conjugation reaction in conjunction with CDMS to demonstrate that modified TMV proteins do not redistribute between disks. Finally, this technique was used to discriminate between protein complexes of near-identical mass but different configurations. We have gained a greater understanding of the behavior of TMV, a protein used across a broad variety of fields and applications, in the solution state.

Accurate Sizing of Nanoparticles Using a High-Throughput Charge Detection Mass Spectrometer without Energy Selection

“…Although the stochastic nature of particle charging inherently leads to fairly broad charge distributions, the width of a charge distribution within a particular mass range in CDMS analysis of nanoparticles can also provide useful information about the extent of morphological homogeneity in a sample. A bimodal charging distribution has been observed previously in the CDMS analysis of tobacco mosaic virus disk complexes where two disks linked in an edge-on conformation exhibited a charge state distribution above the Rayleigh limit, while a more compact stack of two disks exhibited charge states lower than the Rayleigh limit . It is worth noting that measuring both mass and charge directly using CDMS makes identification of nanoparticle dimers straightforward.…”

“…A bimodal charging distribution has been observed previously in the CDMS analysis of tobacco mosaic virus disk complexes where two disks linked in an edgeon conformation exhibited a charge state distribution above the Rayleigh limit, while a more compact stack of two disks exhibited charge states lower than the Rayleigh limit. 24 It is worth noting that measuring both mass and charge directly using CDMS makes identification of nanoparticle dimers straightforward. In contrast, identification of dimers by scattering or mobility-based methods is more challenging because the signals from dimers are indistinguishable from those of larger single particles.…”

“…In contrast, masses of individual ions are determined in CDMS via independent, simultaneous measurements of both m / z and charge, making it possible to eliminate spectral congestion. − Similar to microscopy-based sizing methods, many individual ion measurements are compiled to yield a mass histogram that is representative of the range of molecules, molecular complexes, or particles in the sample. This approach to weighing ions makes it possible to measure mass distributions even when mixtures or other intrinsically heterogeneous samples are analyzed. ,− CDMS has been used to characterize a wide variety of large biomolecular assemblies, such as viruses and virus-like particles. ,− CDMS has also been used to analyze polymer-based nanoparticles, but these studies had significant instrumentation-based limitations in either mass accuracy or the speed of data acquisition. , …”

Protein-Based Model for Energy Transfer between Photosynthetic Light-Harvesting Complexes Is Constructed Using a Direct Protein–Protein Conjugation Strategy

“…cpTMV assembles into double-layered disks with a hollow pore, with 17 monomers per disk and 34 monomers per assembly, and its assembly state is stable across a broad pH and ionic strength range . Mutants and conjugates of cpTMV have been shown to maintain their assembly state after storage for several days at room temperature . The structure of cpTMV resembles the structure of light-harvesting complexes such as LH1 and LH2, which also assemble into flattened disks composed of identical monomers in circular arrays.…”

“…21 Mutants and conjugates of cpTMV have been shown to maintain their assembly state after storage for several days at room temperature. 22 The structure of cpTMV resembles the structure of light-harvesting complexes such as LH1 and LH2, which also assemble into flattened disks composed of identical monomers in circular arrays. cpTMV has previously been used as a tunable scaffold for the attachment of circular chromophore arrays, with varying constraints and interchromophore distances, to mimic single LHCs, providing insights into the physical characteristics underpinning photosynthetic energy transfer.…”