Award details

Tag & Charge A new approach to simultaneously enrich and enhance phosphoproteome analysis

Reference	BB/I012354/1
Principal Investigator / Supervisor	Professor William Griffiths
Co-Investigators / Co-Supervisors	Professor Yuqin Wang
Institution	Swansea University
Department	Institute of Life Science Medical School
Funding type	Research
Value (£)	285,429
Status	Completed
Type	Research Grant
Start date	01/03/2012
End date	28/02/2015
Duration	36 months

Abstract

In a typical proteomics workflow, proteins are digested with trypsin and the resultant peptides separated through one or two dimensions of liquid chromatography and analysed by mass spectrometry (MS). Because phosphopeptides are normally present at low stoichiometry, and also the negatively charged phosphate group attenuates their ionisation efficiency during MS analysis, phosphopeptides are often masked by non-phosphopeptides and are notoriously difficult to detect. Therefore, in phosphoproteomic studies it is essential to enrich for phosphopeptides when working with complex mixtures. In this proposal we will utilise a chemical derivatisation strategy for (i) the specific enrichment of phosphopeptides and (ii) their improved analysis by MS. This will be achieved by the utilisation of click chemistry, where a phosphopeptide is initially derivatised through a phosphoramidate bond with an alkyne tag which is 'clicked' to an azide-linked photocleavable biotin reagent. The resultant biotin linked phosphopeptides can then be enriched on avidin beads (while contaminating peptides are washed away) and then released from the beads by a photo-cleavage. The photo-cleavage will result in an amine linked triazole bound through the phosphoramidate bond to the phosphate group of the phosphopeptide. This effectively results in charge reversal, converting the acidic amino acid residue to one which is basic. The consequence is improved MS response of the derivatised phosphopeptides and the generation of signature fragment-ions in the resultant CID spectra. Thus, a new strategy for phosphoproteomics will be developed offering an improved method for phosphopeptide enrichment and MS analysis leading to deeper mining of the phosphoproteome.

Summary

Reversible protein phosphorylation has turned out to be a general control mechanism involved in almost all aspects of cellular process. The significance and complexity of reversible protein phosphorylation can be assessed from the fact that there are at least 518 protein kinases and more than 100 protein phosphatases present in the human genome, and it is estimated that one third of proteins are phosphorylated in a given cellular proteome. Dysregulation of protein phosphorylation signalling pathways is a major factor leading to different types of human disease, including cancers. Currently about 30% of all drug development programs across the pharmaceutical industry are focused on kinase inhibitors. So far only the skeleton of the body of protein phosphorylation pathways has been uncovered and the interaction maps between individual kinases, phosphatases and their substrates are far from clear. Despite major advances in phosporylation site discovery, utilizing enrichment strategies in combination with mass spectrometry, a major part of the phosphoproteome still remains undiscovered. In this proposal we seek to open a new window on the phosphoproteome by developing new methods for phosphopeptide enrichment and analysis by mass spectrometry, there by allowing the detection of previously undetected phosphorylation sites. As protein phosphorylation is one of the most important mechanisms for controlling cellular process, and kinase and phosphatase inhibitors are the focus of much pharmaceutical interest, the outcome of this study will benefit a wide range of investigators from both academic and pharmaceutical sectors. In the long term it will also benefit patients whose illness is a consequence of dysregulation of protein phosphorylation. In the short term the chemistry developed has the potential for commercial 'packaging' in a kit format.

Impact Summary

Who will benefit from this research? This project is to develop new tools for the systematic analysis of protein phosphorylation. As protein phosphorylation is one of the most important mechanisms for controlling cellular process, and kinase and phosphatase inhibitors are the focus of much pharmaceutical interest, the outcome of this study will benefit a wide range of investigators from both academic and pharmaceutical sectors. In the long term it will also benefit patients whose illness is a consequence of dysregulation of protein phosphorylation. In the short term the chemistry developed also has the potential for commercial 'packaging' in a kit format. How will they benefit from this research? The developed technology will offer a number of advantages over current phosphopeptide enrichment methods. We believe it, in combination with existing proteomics platforms, will enable the discovery of new phosphorylation events. The community can apply these new tools in quantitative phosphoproteomics studies, comparing normal and disease samples, to find out the key points of dysregulation which lead to disease, e.g. in cancer, inflammation and Alzheimer's disease. This could lead to the development of new therapeutics. The new tools can be used to evaluate the specificity of recently developed kinases inhibitors using a systematic approach, which will be particular useful to the pharmaceutical industry. The RA working on this project will gain a wide range of experience in the areas of protein chemistry, micro-derivatisation chemistry, mass spectrometry and bioinformatics. In particular, the RA will benefit from the multidisciplinary aspect of this project by applying chemical tools to solve biological problems and working in an exciting and productive environment. What will be done to ensure that they benefit from this research? We are an active group in terms of publication and participation at international conferences, and have strong links to the UK pharmaceutical industry. WJG is regularly invited as a plenary speaker to international conferences. Over the past 12 months he has given invited lectures at meetings in the USA, Japan, Austria, Hungary, and in the UK. The results of this study will be published in peer reviewed journals and we will also submit the identified phosphorylation sites with mass spectra to public databases such as PhosphoELM. We are collaborating with Prof Dean Nizetic from Queen Mary, University of London, on a phosphoproteomics project entitled 'Interacting regulons of NRSF/REST and DYRK1A in neuron generation, survival and renewal by ultra-sensitive proteomics', and it is likely that the developed technology will be utilized in that project. There is also an opportunity for commercialisation of the developed technology into a kit format. We are already collaborating with a commercial company in this regard with respect to other derivatisation methods. The University is equipped with a department to manage commercial exploitation of research.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Technology and Methods Development
Research Priority	Technology Development for the Biosciences
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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