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Exploring the interplay of dynamic protein modifications in human (patho) physiology


   Institute of Systems, Molecular and Integrative Biology

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  Prof Claire Eyers, Prof A Jones, Prof Gary Kruppa  No more applications being accepted  Funded PhD Project (UK Students Only)

About the Project

Proteins are extensively regulated by dynamic, often reversible post-translational modifications (PTMs). This process allows cells to respond rapidly to environmental factors, be that e.g. growth factors, stressors, or contact adhesion with other cells, ultimately facilitating an intracellular response tailored to the specific extracellular stimuli and allowing cells to adapt. Importantly, protein modifications have been shown to be differentially regulated in numerous diseases, including cancer, serving as both markers of diseases and facilitating the understanding of disease progression.

Although over 400 biologically relevant PTM types have been reported, in mammalian systems much of the effort in PTM characterisation has been focussed on the phosphorylation of serine, threonine, and tyrosine residues, as well as lysine ubiquitination, methylation/acetylation of lysine and arginine residues, and protein glycosylation. We have recently demonstrated that non-canonical phosphorylation of 6 other amino acids is extensive in human cells, and developed mass spectrometry-based strategies to explore other understudied PTMs including sulfation and redox status. This project, which is a collaboration between academics at the University of Liverpool and Bruker Daltonics, seeks to further develop these analytical pipelines for exploration of PTM dynamics and interplay in human (patho)physiology.

While primarily being a technological programme advancing state-of-the-art MS proteomics capabilities for dynamic PTM analysis, this studentship will build on the interests and expertise of the primary and secondary supervisors in cell signalling biology, proteomics-based method development and computational biology, who have a demonstrable track record of collaboration. While we will use this technology primarily to explore PTMs dynamics of human proteins, the technologies and pipelines developed will be applicable to all species, given the fundamental importance of protein modifications to all areas of biological signalling.

Specific objectives:

1.      Develop an in-house system for sensitive ion mobility-based separation of (near) isobaric peptide analytes using the timsTOF Pro mass spectrometer, implementing PASEF (parallel accumulation and serial fragmentation) for discrimination of e.g. phosphorylated and sulfated peptides, and enhancing separation of isomeric analytes (i.e. where the same peptide sequence contains the same PTM type at different sites).

2.      Explore real-time data searching for the enhanced identification of peptides carrying PTMs.

3.      Combine enhanced analytical capabilities (from 1.) with predicted PTM space following e.g. Open PTM searches, to develop a strategy for research-led intelligent PTM detection.

4.      Apply the developed pipelines to explore and quantify PTM interplay in U2OS cells exposed to cellular stimuli/stressors/inhibitors, including regulators of protein kinase networks and mitochondrial dysfunction.

5.      Exploit a variety of computational strategies e.g. residue conservation, motif discovery, pathway analysis, mining protein-protein interaction databases to cross-correlate PTM types and sites and predict interplay and regulatory mechanisms.

Outcomes:

·        New SOPs (standard operating procedures) for site-specific identification of protein PTMs using an advanced analytical and computational framework, that can be shared with the biological and systems biology communities.

·        Understanding of the extent of PTMs on human proteins, and their contextual information

·        Application notes and publications in good journals, in terms of the analytical and computational methods developed but also understanding PTM interplay in response to cellular perturbation.

·        Opportunities to understand industrial priorities and drivers

·        Experience in presentation of findings at appropriate analytical/technology and cell signalling meetings

·        Training in fundamental MS and computational biology, making future employability high

The proposed start date is October 2022

For any enquiries please contact Professor Claire Eyers on: [Email Address Removed] 

To apply for this opportunity, please send a CV and covering letter to Professor Claire Eyers on: [Email Address Removed] 


Funding Notes

This is a CASE PhD Studentship funded by Bruker Daltonics

References

• Daly LA, Brownridge PJ, Batie M, Rocha S, See V* and Eyers CE* (2021) Oxygen-dependent changes in HIF binding partners and post-translational modifications regulate stability and transcriptional activity. Science Signaling (2021) 14 (692), eabf6685
• Hardman G, Perkins S, Brownridge P, Clarke CJ, Byrne DP, Campbell AE, Kalyuzhnyy A, Myall A, Eyers PA, Jones A, Eyers CE*. Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation. The EMBO J. (2019) e100847. doi.org/10.15252/embj.2018100847
• Lanucara F and Eyers CE*. Top-down Mass Spectrometry for Discovery of Combinatorial Post-translational modifications. Mass Spectrometry Reviews (2012) 32:27-42. doi: 10.1002/mas.21348
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