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Department of Biochemistry

 
lilley

Cambridge Centre for Proteomics

 

 CCP NEWS:

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CCP welcomes a new post-doctoral researcher, Benedict Dirnberger. who will be working on a joint project with the Russell group in the Department of Genetics

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Congratulations to all lab members involved in Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS) paper just published by Nature Biotechnology.

for more details see:

www.nature.com/articles/s41587-018-0001-2

and

naturemicrobiologycommunity.nature.com/badges/292-contributor/posts/42434-new-tricks-with-an-old-method

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CCP Overview

The Cambridge Centre for Proteomics (CCP) is comprised of two groups of which Kathryn Lilley is the director, a research group  and a core facility that provides facilities for the School of the Biological Sciences and for wider collaborators in Cambridge and the UK. CCP offers a fee for service proteomics resource.

 

CCP Research

We develop technologies which enable measurement of the dynamics of the proteome in a high throughput manner in space and time during cellular processes such as signalling and differentiation.

1. Protein location

Localisation of Organelle Proteins using Isotope Tagging (hyperLOPIT) (1,2), enables the assignment of proteins and protein complexes to sub-cellular locations, has been applied successfully to several biological systems (3,4). The ability to assign individual proteins accurately to specific sub-cellular structures and monitor their movement within cells is of paramount importance to our understanding of cellular mechanisms.  hyperLOPIT, is being applied in many collaborations including Martinez Arias (Genetics) and applied to various projects including the Oliver, Griffin, Howe and Waller groups (Biochemistry) and Martinez Arias (Genetics),  Smith (UCL), Larsen (Odense), Lundberg (KTH Royal Institute of Technology). We have also extended the LOPIT workflow repertoire - bioRxiv 378364 -  https://doi.org/10.1101/378364

2. RNA location and the RNA binding proteome

We are also developing methods to map the location of the transcriptome in collaboration with Anne Willis (MRC Toxicology Unit, Leicester). This project is funded by a Wellcome Trust Joint Investigator award (110170/Z/15/Z).(19.

We have devised a very efficient method to enrich the RNA binding proteome, which allows both characterisation of RBPs (RNA bound proteins) and PBRs (protein bound RNA). The method, OOPS, (orthogonal organic phase separation), is based on UV cross-linking and aqueous/organic phase separation using Trizol. For more details see

3. Multi protein complexes

i. Protein protein interaction methods. Interactomes using Parallel Affinity Capture (iPAC) is a method developed in collaboration with the St Johnston (Gurdon Institute) and Russell (Genetics Dept.) groups to determine genuine residents of multi protein complexes (5). This method is being applied to a variety of different biological systems (6,7).

ii. Selective Proteomic Proximity Labelling using Tyramide (SPPLAT). Developed in collaboration with the Tony Jackson (Biochemistry) and Sarah Perrett (University of Chinese Academy of Sciences), this method enables the identification of proteins in the immediate vicinity of a target membrane protein (8).

4. Quantitative methods

Development of quantitative proteomics workflows involving label free methods (9, 10) and isobaric tagging  (11, 12)

5. Computational proteomics

Robust statistical and computational data analysis is of vital importance to the above techniques, and to proteomics in general, to ensure that data sets are efficiently mined and do not contain unacceptable levels of false discovery. CPU comprises a dedicated team of informaticians to develop bioinformatics and statistical tools, that utilize pattern recognition and machine learning methods to enable robust analysis of organelle proteomics and multi-protein complex data (13,14). The output of this research is manifested in the creation of open-source software solutions for quantitative data analysis that are applicable to the majority of quantitative proteomics applications (15).

 

Applications of technologies:

1. DDIP - Drosophila developmental interactome project. We are working as part of a  large study across the Kathryn Lilley, Steve Russell (Genetics) and Alfonso Martinez Arias (Genetics) groups in Cambridge and colleagues at the University of Manchester (Simon Hubbard) and University College London (David Jones and Christine Orengo), to map the alternatively spliced proteome during the early stages of Drosophila development in a quantitative manner, and to look at its impact on protein protein interactions, sub cellular localization and protein structure, with emphasis on components of major signaling pathway proteins (16). This study is funded by a BBSRC sLoLa.

2. Detox - CCP is part of a consortium with Gavin Thomas and Tony Larsen (York), Gill Stephens (Nottingham), Dave Kelly and Jeff Green (Sheffield)  Susan Molyneaux Hodgson (Exeter) and   Lucite, Green Biologics, Ingenza and the CPI. The project aims to provide the first ever systematic analysis of how chemicals poison bacterial cells. http://projectdetox.co.uk/. This work in funded through a BBSRC IB Catalyst award. (17)

 

 

Lab members:  Anja Andrejeva (Core facility) Lisa Breckels, Konstantin Barylyuk (Waller lab), Josie Christopher, Olly Crook, Michael Deery (Core facility. manager), Benedict Dirnberger, Mo Elzek, Renata Feret (Core facility), Aikaterini Geladaki, Julie Howard (Core facility),  Kathryn Lilley (Director of CCP), David-Paul Minde, Mie Monti (MRC Toxicology Unit, Leicester), Daniel Nightingale,  Rayner Queiroz, Manasa Ramakrishna (MRC Toxicology Unit, Leicester), Lise Skov, Tom Smith, Yagnesh Umrania (Core facility), Owen Vennard, Eneko Villanueva, Hongtao Zhang (Rothamstead)

 

Visit Cambridge Proteomics Centre website

 

Key publications:

1. Queiroz RML,  Smith T, Villanueva et al (2019) Comprehensive quantitation of RNA-protein interaction dynamics by orthogonal organic phase separation (OOPS), Nature Biotechnology  doi:10.1038/s41587-018-0001-2

2.Harvey RF, et al. (2018) Trans-acting translational regulatory RNA binding proteins. Wiley Interdiscip Rev RNA. 2018 May;9(3):e1465. doi: 10.1002/wrna.1465

3. Nightingale DJH, Geladaki A, Breckels LM, Oliver SG, Lilley KS (2018) The subcellular organisation of Saccharomyces cerevisiae. Current Opinions in Chemical Biology, doi.org/10.1016/j.cbpa.2018.10.026

4. Crook OM, Mulvey CM, Kirk PDW, Lilley KS, Gatto L.A Bayesian mixture modelling approach for spatial proteomics. PLoS Comput Biol. 2018 Nov 27;14(11):e1006516. doi: 10.1371/journal.pcbi.1006516

5. Mulvey,CM et al (2017) Using hyperLOPIT to perform high resolution mapping of the spatial proteome. Nature Protocols. Jun;12(6):1110-1135. doi: 10.1038/nprot.2017.026

6. Thul P et al (2017) A subcellular map of the human proteome Science.  May 26;356(6340). pii: eaal3321. doi: 10.1126/science.aal3321

7. Christoforou et al (2016)  A draft map of the mouse pluripotent stem cell spatial proteome. Nature Communications  Jan 12;7:8992 doi:10.1038/ncomms9992

8.  Minde D-P, Dunker AK and Lilley KS (2017) Time, space, and disorder in the expanding proteome universe. Proteomics 17(7). doi: 10.1002/pmic.201600399

9. Lowe N, et al. (2014) Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library. Development 141(20):3994-4005. doi: 10.1242/dev.111054.

10. Chen C, et al  (2015) Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature  Nature  Nov 18. doi: 10.1038/nature16148

11. Li XW, et al (2014) New insights into the DT40 B-Cell Receptor cluster using a proteomics proximity labelling assay. J. Biol. Chem.;289(21):14434-47. doi: 10.1074/jbc.M113.529578

12. Vowinckel, J et al (2016) The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics.doi: 10.12688/f1000research.2-272.v2. eCollection 2013.

13. Mulvey CM, et al (2015) Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells. Stem Cells. 2015 Sep;33(9):2712-25. doi: 10.1002/stem.2067. Epub 2015 Jun 23.

14. Breckels, LM, et al (2016) Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics. PLoS Comput Biol. 2016 May 13;12(5):e1004920. doi: 10.1371/journal.pcbi.1004920.

15. Gatto L, et al (2014) Mass-spectrometry based spatial proteomics data analysis using pRoloc and pRoloc data. Bioinformatics. 30 (9) 1322-4

 16. Fabre, BF, et al (2016) Analysis of the Drosophila melanogaster proteome dynamics during the embryo early development by a combination of label-free proteomics approaches. Proteomics. 2016 Mar 31. doi: 10.1002/pmic.201500482.

17.Marondedze, C et al, (2016) The RNA-binding protein repertoire of Arabidopsis thaliana.  Scientific Reports 11;6:29766. doi: 10.1038/srep29766