Development of novel mass spectrometry approaches that enable phosphoproteomic analysis in tandem with cellular localisation

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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).