Dynamics and interplay of protein modifications

This collaborative application requests support for provision of a sensitive, high resolution, versatile, state-of-the-art mass spectrometry platform to drive cutting edge proteomics research. The applicants are all major contributors to the UK science infrastructure through research, training and services rendered, and as scientists whose research fundamentally relies on cutting edge proteomics, recognise the pressing need to increase capacity and drive state-of-the-art proteomics capabilities in the North West. The equipment requested, specifically a Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer (Fig. 1) with associated ultra-high performance liquid chromatography (UHPLC) separation system, will be based in the internationally-renowned Centre for Proteome Research (CPR) at the University of Liverpool (UoL). The unique features and capabilities of the Ascend will enhance fundamental and applied UKRI collaborative research and fee-for service offerings, both at UoL, and across the UK. This instrument will be used for state-of-the-art structural investigation and quantification of protein post-translational modifications (PTMs), notably the addition of functional groups (small chemical moieties, proteins, sugars, or lipids) that induce a rapid change in protein structure and thus alter biological function. Such PTMs play diverse roles in cell signalling, both as individual entities and in combination, often on the same protein, and regulate all aspects of biology across the BBSRC remit. Precisely defining and quantifying changes at the single site level and as PTM ‘fingerprints’ arising due to system perturbation is thus essential to revolutionise biological understanding. As well as advancing capabilities by nature of its design, this platform will also enhance much needed capacity in Liverpool and regionally, complementing aging instrumentation within the CPR where it will be housed and maintained. Crucially, access to this technology is extremely limited in the UK and will therefore open new research possibilities for academics in Liverpool and across the North, as well as industry and clinical partners. Aims Methodological developments for the site-specific and combinatorial analysis of labile, difficult and uncommon PTMs Capability enhancement for relevant UoL BBSRC-funded research projects Capacity building across the UK in PTM analysis Objectives Addressing our aims will: Build on our pioneering work in the development of novel proteomics methodologies for the analysis of challenging PTMs, advancing sensitive analytical strategies for the characterisation and quantification of e.g. i) non-canonical phosphorylation; ii) Tyr-sulfation and nitration; iii) Cys-based oxidation; iv) hydroxylation; v) nucleotidylation; vi) PTMs at the single cell level (single-cell proteomics, SCP). As well as developing peptide-level approaches, we will also (where relevant) expand our suite of tools for protein (proteoform) investigation to better understand the role of combinatorial modifications (PTM ‘fingerprints’). Open up capabilities in PTM analysis for BBSRC (and other UKRI)-funded projects in Liverpool and across the NorthWest relevant to human, animal and environmental health, exemplars of which are included below. Work with, and train, end-users, in line with our role as part of the Liverpool Shared Research Facilities (LIV-SRF, see below), and key role delivering the NERC environmental multi ‘omics training facility, to build UK-wide capacity in the characterisation and quantification of protein PTMs.