Insights into virus capsid assembly from non‐covalent mass spectrometry

Morton, Victoria L.; Stockley, Peter G.; Stonehouse, Nicola J.; Ashcroft, Alison E.

doi:10.1002/mas.20176

Cited by 48 publications

(33 citation statements)

References 109 publications

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“…Nano-ESI is a mild ionization technique that preserves non-covalent interactions and is therefore suitable for the analysis of protein conformation or supramolecular complexes [62]. The capability to preserve non-covalent interactions and the strong analytical power of mass spectrometry make nano-ESI a powerful tool for intact protein analysis [63,64]. Fig.…”

Section: Spaap Dimerizationmentioning

confidence: 99%

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

Papaleo

Parravicini

Grandori

et al. 2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Section: Spaap Dimerizationmentioning

confidence: 99%

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

Papaleo

Parravicini

Grandori

et al. 2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…Thus, rather than an absolute measurement, a relative exponential correlation is generated between the measured drift times and Ω 1,13 : where t D is the measured drift time, and X is the proportion constant that can be extracted from a calibration curve. The calibration is performed by measuring the drift times of ions with known Ω (measured from conventional IM experiments).…”

Section: Part 3: Correlating Between Drift Time Values and Cross-sectmentioning

confidence: 99%

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Michaelevski

Kirshenbaum

Sharon

2010

JoVE

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Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N 2 ) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (Ω).Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as Ω is related to the overall shape and topology of the ion. The correlation between the measured drift time values and Ω is calculated using a calibration curve generated from calibrant proteins with defined cross-sections , successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available) 10 . We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical Ω values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field. Video LinkThe video component of this article can be found at https://www.jove.com/video/1985/ ProtocolThe procedure we describe focuses solely on IM-MS analysis of protein complexes. Therefore, we suggest that researchers unacquainted with the field of structural MS refer to the sample preparation steps, instrument calibration and MS and tandem MS optimization procedures described in Kirshenbaum et al. 2009 http://www.jove.com/index/details.stp?ID=1954. In general, this protocol involves low micromolar concentrations of complex (1-20 μM) in a volatile buffer such as ammonium acetate (0.005 -1 M, pH 6-8). Given that 1-2 μl are consumed per nanoflow capillary, we suggest 10-20 μl as a minimum volume, to enable optimization of MS conditions. Part...

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“…In these studies, sufficient mass resolution was obtained to determine the accurate mass and stoichiometry of the T ϭ 3 and T ϭ 4 HBV capsids despite their large mass of 3 and 4 million Da, respectively (15,17). In addition to being able to measure the mass and stoichiometry of protein assemblies, the capacity of native MS to analyze simultaneously a heterogeneous population of assembly intermediates makes it a powerful technique to study virus assembly (27).…”

mentioning

confidence: 99%

Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

Shoemaker

Duijn

Crawford

et al. 2010

Molecular & Cellular Proteomics

124

123

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Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T ‫؍‬ 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1 60 (3.36-MDa) and VP1 80 (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1 180 ). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T ‫؍‬ 3 VP1 180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1 60 and VP1 80 species. Molecular & Cellular Proteomics 9: 1742-1751, 2010.

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Insights into virus capsid assembly from non‐covalent mass spectrometry

Cited by 48 publications

References 109 publications

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

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