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

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Wang Y (2014) 24S,25-Epoxycholesterol in mouse and rat brain. in Biochemical and biophysical research communications

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William Griffiths (Co-Author) (2013) Identification of LXR interacting proteins

 
Description This project was targeted at derivatisation of phosphate groups for subsequent phosphopeptide analysis. A "bonus" finding was an improved method to methylate carboxylic acid groups. In the project we were able to effectively convert phosphate groups on peptides to phosphoramidates linked to an alkyne function and then successfully "click" the alkyne to an immobilised azide, their by allowing phosphopeptide pull down for subsequent mass spectrometry analysis. However, when used for complex mixtures of peptides the efficiency of the derivatisation chemistry was variable making application to biological samples difficult and in some cases non-reproducible. More work will be required to improve the derivatisation chemistry.

The bonus finding has proved particularly useful as derivatisation with trimethylsilyldiazomethane has proved specific for carboxylic acids and avoids methylation of the enol forms of carbonyls, allowing the simple differentiation of carboxylic acids from molecules with both a carbonyl and alcohol by mass spectrometry.
Exploitation Route The derivatisation methods developed may have wide applications in proteomics, lipidomics and metabolomics.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Although the project was targeted at derivatisation towards phosphate groups, a spin-off discovery was an improved method to methylate carboxylic acid groups. This is now being explored by us in collaboration with third sector organisations and in the development of "kit technology" for derivatisation.
First Year Of Impact 2014
Sector Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Mass Spectrometry Based Lipidomics and Metabolomics to Drive Bioscience Discovery
Amount £748,381 (GBP)
Funding ID BB/S019588/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 06/2020
 
Title Acids and Ketones 
Description Using mass spectrometry it is a challenge to differentiate the combination of an alcohol group and a carbonyl from a carboxylic acid as both add 29.9742 u to the core structure. One solution we uncovered in this work is derivatisation with trimethylsilyldiazomethane which specifically methylates the acid but not the enol form of the carbonyl. A common strategy in mass spectrometry is derivatisation of carbonyl groups with amines or hydrazines to form oximes or hydrazones. These reactions proceed to completion with alpha,beta-unsaturated ketones but not saturated ketones. We have been able to improve the derivatisation yield with saturated ketones by performing the reaction in acidic methanol rather than the usual aqueous acidic methanol solvent. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? No  
Impact A book chapter describing these improved derivatisation methods is in preparation. These improved methods are currently used in our group e.g. in PMID: 30736477 
 
Description Cardiff Neuroscience 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysis of cultured cells for oxysterols
Collaborator Contribution Provision of cellular material
Impact Manuscript in preparation
Start Year 2014
 
Description ENOR 
Organisation Catholic University of Louvain
Country Belgium 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation Minerva Foundation Institute for Medical Research
Country Finland 
Sector Private 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University Hospital Regensburg
Country Germany 
Sector Hospitals 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University of Basel
Country Switzerland 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University of Clermont Auvergne
Country France 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University of Edinburgh
Department Queen's Medical Research Institute Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University of Franche-Comté
Country France 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University of Oslo
Country Norway 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description ENOR 
Organisation University of Toulouse
Country France 
Sector Academic/University 
PI Contribution expertise, intellectual input, access to equipment
Collaborator Contribution Access to biological materials
Impact doi: 10.1016/j.jsbmb.2019.03.02 doi: 10.1016/j.biochi.2018.07.016
Start Year 2010
 
Description Edin 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided intellectual input and data.
Collaborator Contribution Edinburgh has provided intellectual input and data.
Impact Paper in Immunity and PLoS Biology
Start Year 2012
 
Description Lipidomics Standards Initiative 
Organisation Babraham Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Lipidomics Standards Initiative 
Organisation Cardiff University
Department School of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Lipidomics Standards Initiative 
Organisation Medical University of Graz
Country Austria 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Lipidomics Standards Initiative 
Organisation University of Pardubice
Country Czech Republic 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Lipidomics Standards Initiative 
Organisation University of Regensburg
Country Germany 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Lipidomics Standards Initiative 
Organisation University of Southern Denmark
Country Denmark 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Lipidomics Standards Initiative 
Organisation University of Texas Southwestern Medical Center
Country United States 
Sector Academic/University 
PI Contribution Intellectual input to the Lipidomics Standards Initiative.
Collaborator Contribution Intellectual input.
Impact doi.org/10.1038/s42255-019-0094-z
Start Year 2018
 
Description Université de Bourgogne-Franche Comté 
Organisation University of Franche-Comté
Country France 
Sector Academic/University 
PI Contribution expertise, intellectual input, data
Collaborator Contribution access to biological materials
Impact doi: 10.1016/j.biochi.2018.02.008.
Start Year 2017
 
Description BBC Wales A Healthy Future 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact BBC Radio Wales described our mass spectrometry technology and its value for disease diagnosis and discovery of novel therapeutics.
Year(s) Of Engagement Activity 2019
URL https://www.bbc.co.uk/programmes/m0007yzp