Abstract
Gas-phase electrophoretic mobility molecular analysis (GEMMA) separates nanometer-sized, single-charged particles according to their electrophoretic mobility (EM) diameter after transition to the gas-phase via a nano electrospray process. Electrospraying as a soft desorption/ionization technique preserves noncovalent biospecific interactions. GEMMA is therefore well suited for the analysis of intact viruses and subviral particles targeting questions related to particle size, bioaffinity, and purity of preparations. By correlating the EM diameter to the molecular mass (Mr) of standards, the Mr of analytes can be determined. Here, we demonstrate (i) the use of GEMMA in purity assessment of a preparation of a common cold virus (human rhinovirus serotype 2, HRV-A2) and (ii) the analysis of subviral HRV-A2 particles derived from such a preparation. (iii) Likewise, native mass spectrometry was employed to obtain spectra of intact HRV-A2 virions and empty viral capsids (B-particles). Charge state resolution for the latter allowed its Mr determination. (iv) Cumulatively, the data measured and published earlier were used to establish a correlation between the Mr and EM diameter for a range of globular proteins and the intact virions. Although a good correlation resulted from this analysis, we noticed a discrepancy especially for the empty and subviral particles. This demonstrates the influence of genome encapsulation (preventing analytes from shrinking upon transition into the gas-phase) on the measured analyte EM diameter. To conclude, GEMMA is useful for the determination of the Mr of intact viruses but needs to be employed with caution when subviral particles or even empty viral capsids are targeted. The latter could be analyzed by native MS.
Highlights
condensation particle counter (CPC) is exclusively based on the number of particles practically irrespective of their molecular mass (Mr) or chemical nature
It is of note that only advancements in Gas-phase electrophoretic mobility molecular analysis (GEMMA) instrumentation of the past decade allowed for the differentiation of intact HRVA2 and the contaminant because previous setups only yielded one broad peak at the electrophoretic mobility (EM) diameter of intact virus including both species
A2 of high purity, (ii) to check its behavior in comparison to virus preparations obtained via our conventional conditions, and (iii) to employ this highly pure HRV-A2 for GEMMA and native MS measurements, both based on an electrospray process, for the establishment of a Mr/EM diameter calibration curve applicable for virus Mr determination
Summary
CPC is exclusively based on the number of particles practically irrespective of their molecular mass (Mr) or chemical nature. HRV-A2 (a HRV genus A serotype 2) is a nonenveloped icosahedral virus of approximately 30 nm diameter as based on X-ray crystallography,[28] cryo-electron microscopy 3D image reconstruction (cryo-EM 3DR),[29] and previous GEMMA3 measurements It is composed of four viral proteins (VP1−4), 60 copies each, and a single stranded, positive sense RNA genome of approximately 7.1 kb length, covalently linked to a single copy of the peptide VPg.[30,31] During virus uncoating (i.e., the process of viral RNA transfer from the capsid into the cytosol of an infected cell), the protein shell undergoes conformational changes resulting in subviral particles; the intermediate A-particle has lost VP4 but still contains the RNA genome. The correlation established here between EM diameter and Mr for intact, spherical, native viruses allows one to determine, via GEMMA, the Mr of viruses in general with greater precision than has been possible to date
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