Development of novel mass spectrometry approaches that enable phosphoproteomic analysis in tandem with cellular localisation
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
As a key mediator of cellular signalling, phosphorylation remains a principal target for biological question. Identifying and quantifying the phosphorylation state of proteins involved in cell progression, metabolism, growth and disease is critical for the elucidation of cellular function. The initial aim of the project with be to extend recent developments that have demonstrated that the combination of phospho-peptides enrichment using titanium matrices and isobaric chemical labelling (TMT) allows the identification of 10,000 to 15,000 phosphopeptides from total cell lysate (1, 2). This coupled with the hyperLOPIT methodology (Localisation of Organelle proteins by isotope tagging, (3-5), which combines a biochemical organelle fractionation with a quantitative mass spectrometry analysis, will enable proteomic analysis at the organelle level. Whilst scientifically and experimentally challenging it is realistic to envisage the combination of these methods to deliver a novel approach which will add cellular resolution to phosphoproteomic analysis.
The second goal of the research will be the application of these methods to probe disease relevant cell types and response to compound treatment. This will leverage AstraZeneca's disease expertise and wealth of annotated probe compounds. Hypotheses generated from these analyses with be further tested by, targeted proteomic strategies (Selected Reaction Monitoring) and antibody methods to directly characterise the phosphorylation state of the protein candidates and their localisation within the cell using fluorescence microscopy. Finally, exploiting AstraZeneca's access to disease relevant tissues, the expression of targeted protein candidates can be validated on mouse/human samples.
(1) Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer. Erickson BK, Jedrychowski MP, McAlister GC, Everley RA, Kunz R, Gygi SP. Anal Chem. (2015).
(2) Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells. Melo-Braga MN, Schulz M, Liu Q, Swistowski A, Palmisano G, Engholm-Keller K, Jakobsen L, Zeng X, Larsen MR. Mol Cell Proteomics. (2014).
(3) A draft map of the mouse pluripotent stem cell spatial proteome. Christoforou A, Mulvey CM, Breckels LM, Geladaki A, Hurrell T, Hayward PC, Naake T, Gatto L, Viner R, Martinez Arias A, Lilley KS. Nat Commun. 2016.
(4) Using hyperLOPIT to perform high-resolution mapping of the spatial proteome. Mulvey CM and et al Nature Protocols 12(6):1110-1135. doi: 10.1038/nprot.2017.026 (2017).
(5) A subcellular map of the human proteome. Thul, PJ et al, Science 356(6340). pii: eaal3321. doi: 10.1126/science.aal3321 (2017).
The second goal of the research will be the application of these methods to probe disease relevant cell types and response to compound treatment. This will leverage AstraZeneca's disease expertise and wealth of annotated probe compounds. Hypotheses generated from these analyses with be further tested by, targeted proteomic strategies (Selected Reaction Monitoring) and antibody methods to directly characterise the phosphorylation state of the protein candidates and their localisation within the cell using fluorescence microscopy. Finally, exploiting AstraZeneca's access to disease relevant tissues, the expression of targeted protein candidates can be validated on mouse/human samples.
(1) Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer. Erickson BK, Jedrychowski MP, McAlister GC, Everley RA, Kunz R, Gygi SP. Anal Chem. (2015).
(2) Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells. Melo-Braga MN, Schulz M, Liu Q, Swistowski A, Palmisano G, Engholm-Keller K, Jakobsen L, Zeng X, Larsen MR. Mol Cell Proteomics. (2014).
(3) A draft map of the mouse pluripotent stem cell spatial proteome. Christoforou A, Mulvey CM, Breckels LM, Geladaki A, Hurrell T, Hayward PC, Naake T, Gatto L, Viner R, Martinez Arias A, Lilley KS. Nat Commun. 2016.
(4) Using hyperLOPIT to perform high-resolution mapping of the spatial proteome. Mulvey CM and et al Nature Protocols 12(6):1110-1135. doi: 10.1038/nprot.2017.026 (2017).
(5) A subcellular map of the human proteome. Thul, PJ et al, Science 356(6340). pii: eaal3321. doi: 10.1126/science.aal3321 (2017).
People |
ORCID iD |
| Kathryn Lilley (Primary Supervisor) | |
| Josie Christopher (Student) |
| Description | We have applied the spatial proteomics method to a dynamic system. The dynamic system is irradiating a lung cancer cell line with X-ray to illicit the DNA damage response. We have measured the spatial proteome within this system to investigate protein trafficking between subcellular compartments. Some hits have already been identified, though these need to be validated and undergo more thorough analysis. |
| Exploitation Route | The aim of the project is to explore the hits from this "screening" technique. However, it would be unrealistic to follow up all potential new targets from this high-throughput technique. Therefore, others may be interested to further investigate these. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Determining the content of vesicles captured by golgin tethers using LOPIT-DC |
| Organisation | Medical Research Council (MRC) |
| Department | MRC Laboratory of Molecular Biology (LMB) |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Personally provided technical assistance in performing LOPIT-DC. The research team also provided mass spectrometry and bioinformatic aid. |
| Collaborator Contribution | Performed CRISPR knock-outs, transfections and siRNA knock-down cell lines. Performed confocal microscopy and correlative FM and electron tomography |
| Impact | Publication of the work is in progress. |
| Start Year | 2017 |
| Description | Poster presentation (Adelaide, Australia) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Evoked conversation about the method. Helped to advertise the method and helped to decide how to continue with the project. Potentially opened up to further collaborations. |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://www.hupo2019.org/ |
| Description | Poster presentation (Bradford) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | Sparked questions which aided the project's progress. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Talk at a methods discussion group meeting (St Andrews) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | Gave a talk about the methods developed in our research group at the annual Proteomics Method Forum. This was to both raise awareness of the protocol and discuss the benefits and limitations of the different methods to others in the field - both in academia and industry. This potentially sparked researchers to consider applying the method to their research. |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://www.proteomicsmethodsforum.org.uk/conferences/2019-st-andrews/ |