Advances in mass spectrometry-based proteomics enables comprehensive, quantitative analysis of protein abundance  and protein modification by PTMs like phosphorylation. Analysis of protein-protein interactions in a comprehensive fashion remains challenging. Progress has been made using protein correlation profiling (PCP) , which combines separations of native protein complexes with MS detection. However, a drawback to PCP is that direct evidence for protein-protein interaction is lacking. More recently, researchers have demonstrated that cleavable crosslinkers, such as DSSO, enable the analysis of thousands of crosslinked peptides from a complex cellular extract . Excitingly, detection of these crosslinked peptides provides direct evidence of protein-protein interaction. However, so far direct analysis of crosslinks does not produce the proteome coverage that can be obtained by PCP.
The PhD project aims to develop innovative analytical tools combining PCP and crosslinking analysis by mass spectrometry to characterise protein-protein interactions comprehensively. The methods will be applied to measure the dynamic changes in protein-protein interactions in CD8+ T cell activation. CD8+ T cells are an important class of cells in the adaptive immune response  and function to eliminate cells compromised by viral infection and oncogene activation. In parallel, the student will develop intact mass spectrometry-based methods to analyse the dynamic post-translational modifications and interaction partners of key proteins controlling cellular proliferation, such as Rb. The PhD project aims to answer the following questions about CD8+ T cell activation:
- What are the critical changes in protein-protein interactions?
- What are the dynamics of Rb phosphorylation and protein-protein interactions?
The PhD project provides an exceptional opportunity for cross-disciplinary training, combining analytical development with an application to an important biological system. The Ly laboratory is a dynamic environment with expertise in the areas of analytical chemistry, biochemistry and cell biology. High quality training will be provided on mass spectrometry, facilitated by access to cutting edge instrumentation, including Orbitrap (Eclipse) and FT-ICR mass spectrometers. Analysis of intact proteins isolated from CD8+ T cells will be analysed using intact mass spectrometry with supervision by Dr Dave Clarke (School of Chemistry, University of Edinburgh). The PhD student will be in a unique position in the UK to obtain extensive training and expertise on these instruments and will be leading world-class analytical development using these technologies. Training will be provided on the R scripting language as a tool to obtain biological information from large proteomic datasets.
References:  Kelly et al. https://doi.org/10.1101/2020.07.03.186023,  Kirkwood et al. http://dx.doi.org/10.1074/mcp.M113.032367,  Liu et al. http://dx.doi.org/10.1038/nmeth.3603,  Zhang and Bevan, http://dx.doi.org/10.1016/j.immuni.2011.07.010