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Kavya Clement

Multi-level characterization of bacteriophage T5 capsids and capsid-like particles using mass spectrometry

Published on 24 September 2021
Thesis presented September 24, 2021

Abstract:
Vaccination is the most effective method of infectious disease prevention and control. Current vaccine development strategies do not provide broad access to vaccines in the developing world largely due to high costs and cold chain requirements. Virus Like Particles (VLP) are recombinant viral structures that exhibit immune-protective traits of native virus but are non- infectious. This developing technology has applications in the field of vaccines, gene therapy and antigen display. While most VLPs are derived from human pathogens, viruses infecting bacteria have recently been proposed as promising antigen display platforms. Bacteriophage T5 capsids exhibit 120 copies of a modifiable decoration protein at their surface, and could thus be used as a model for antigen-presenting VLP structures.

The present thesis focuses on studying bacteriophage T5 capsids from different perspectives using multiple mass spectrometry (MS) approaches. One of the aspect is to contribute to an improved understanding of T5 capsid assembly process by determining the stoichiometry of the T5 capsid protease using MS based Proteomics and the other aspect is to assess the integrity and stability of the phage T5 capsids by analyzing their intact mass using Nano-Electro-Mechanical-Sensors Mass Spectrometry (NEMS-MS).

The maturation of the T5 capsid begins with the activation of a protease called pb11, which in turn processes other structural proteins. In spite of its importance for T5 capsid maturation, the exact quantity of pb11 at each stage of the assembly is still subject to debate. The present work describes an original targeted proteomic strategy to determine the presence and copy number of this important protein in mature T5 capsids using a heavy isotope labelled quantification concatemer (QconCAT).

Characterization of massive supramolecular assemblies composed of millions of atoms such as VLPs is very challenging, yet it is an important requirement for vaccine production. NEMS- MS, a novel method for single particle mass sensing, is a promising technology to investigate large biological species that have so far escaped mass characterization. NEMS are nanoscale devices, such as cantilevers or suspended beams, vibrating at their resonance frequency. When a particle lands onto the surface of a NEMS, the device's resonance frequency shifts downwards in proportion to the added mass. Mass sensing can thus be performed by monitoring this frequency in real time. Our study investigates properties independent from particle mass that may influence the uncertainty in mass measurement using NEMS-MS, and how to deal with these issues. An evaluation of the magnitude of all these effects on bacteriophage T5 capsid mass measurements using doubly clamped beams is proposed. Finally, we determine that particle desolvation affects mass measurement more than the physical parameters of the capsid or uncertainties in device geometry.

In conclusion, this work addresses the various attributes and parameters affecting capsid mass determination using nano-resonator-based MS, and reveals the actual copy number of protease protein in mature T5 capsid using nanoLC-MS proteomics.

Keywords:
Bacteriophage T5, Mass spectrometry, NEMS, QconCAT

On-line thesis.