Exploiting and advancing single-cell proteomics to identify novel pathways to disrupt RNA virus infection.

Single-cell studies have been transformative in biology, dominated by single-cell transcriptomics.

Recently, methods for single-cell proteomics by mass spectrometry (SCoPE-MS/SCoPE2) have emerged permitting quantitative analysis of the single-cell proteome.

I will apply SCoPE2 to study RNA virus replication and virus-host interactions with the goal of identifying targets for antiviral intervention.

My preliminary data demonstrates the method has the sensitivity to identify the full range of viral proteins expressed during infection.

To achieve this goal, I propose to advance methods for single-cell proteomics by adapting SCoPE2 for targeted analysis of specific peptides of interest, permitting reproducible sampling of viral peptides.

This proposal has two aims: 1: Develop single-cell proteomics for the analysis of specific viral targets of interest. 2: Employ targeted single-cell proteomics to track changes in viral gene expression at single-cell level. The first aim of this proposal relies on the SCoPE2 method that I helped develop during my postdoctoral research.

By altering the mass spectrometer settings from data-dependent (abundance-based) peptide sampling it is possible to target specific peptides of interest, ensuring reproducibility across multiple experiments. I will optimise SCoPE2 for targeted analysis of RNA virus proteins from single-cell samples.

For aim 2, I will apply the optimised SCoPE2 method developed in aim 1 to perform targeted SCoPE2 analysis of murine norovirus and SARS-CoV-2 infection. Both of these viruses replicate using post-translational cleavage of a long polyprotein.

Targeted protein-level measurements are necessary to track such cleavage at single-cell level, identifying mechanisms of viral replication, and potential targets for antiviral intervention.