Research objectives:
What is the impact of post-transcriptional and post-translational processing of RNA and proteins on their interactions and location?
What are the changes in interactions/location upon perturbation, for example during drug treatment/stress?
How are different proteoforms distributed across subcellular compartments
Do proteins adopt different structures/functions in different locations (moonlighting)?
What role does RNA play in moonlighting (for example RNA-protein interactions and/or localized translation)?
What is the spatial relationship between a protein and its corresponding mRNA?
To enable us to answer the above questions, we have developed a number of technologies and computational tools:
1. Spatial Proteomics
LOPIT - localization of organelle proteomics using isotope tagging / DIA-LOP (label free version of LOPIT)
Technology that provides a map of the subcellular proteome
Geladaki A, Kočevar Britovšek N, Breckels LM, Smith TS, Vennard OL, Mulvey CM, Crook OM, Gatto L, Lilley KS (2019). Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics. Nat. Commun., 10(1):331. doi: 10.1038/s41467-018-08191-w
McCaskie K, Hutchings C, Feret R, Kim YI, Breckels L, Deery M, Lilley K. Localization of Organelle Proteins Using Data-Independent Acquisition (DIA-LOP). Mol Cell Proteomics. 2025 Sep;24(9):101047. doi: 10.1016/j.mcpro.2025.101047.
Sigaeva A, Hutchings C, Cesnik A, Lilley KS, Lundberg E. Subcellular localization as a driver of protein function. Nat Rev Mol Cell Biol. 2026 Feb 18. doi: 10.1038/s41580-026-00947-3
2. RNA binding proteome
OOPS - Orthogonal Organic Phase Separation
An unbiased approach to define the RNA-binding proteome
Villanueva E, Smith T, Queiroz RML, Monti M, Pizzinga M, Elzek M, Dezi V, Harvey RF, Ramakrishna M, Willis AE, Lilley KS (2019). Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS). Nat. Biotechnol., 37(2):169-178. doi: 10.1038/s41587-018-0001-2
Queiroz RML, Smith T, Villanueva E, Marti-Solano M, Monti M, Pizzinga M, Mirea DM, Ramakrishna M, Harvey RF, Dezi V, Thomas GH, Willis AE, Lilley KS (2020) Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS). Nat. Protocols doi:10.1038/s41596-020-0344-2
3. LoRNA - the localisation of RNA
Technology that provides a map of the subcellular transcriptome
Villanueva E, Smith T, Pizzinga M, Elzek M, Queiroz RML, Harvey RF, Breckels LM, Crook OM, Monti M, Dezi V, Willis AE, Lilley KS. System-wide analysis of RNA and protein subcellular localization dynamics. Nat Methods. 2024 Jan;21(1):60-71. doi: 10.1038/s41592-023-02101-94. Protein protein interactions
4. Computational Proteomics
Demichev V, Messner CB, Vernardis SI, Lilley KS, Ralser M (2020). DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods, 17(1):41-44. doi: 10.1038/s41592-019-0638-x
Crook OM, Davies CTR, Breckels LM, Christopher JA, Gatto L, Kirk PDW, Lilley KS. Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE. Nat Commun. 2022 Oct 10;13(1):5948. doi: 10.1038/s41467-022-33570-9
Coleman S, Breckels L, Waller RF, Lilley KS, Wallace C, Crook OM, Kirk PDW. Semi-supervised Bayesian integration of multiple spatial proteomics datasets. PLoS Comput Biol. 2025 Dec 15;21(12):e1013799. doi: 10.1371/journal.pcbi.1013799. PMID: 41396985
Hutchings C, Krueger T, Crook OM, Gatto L, Lilley KS, Breckels LM. An updated Bioconductor workflow for correlation profiling subcellular proteomics. F1000Res. 2025 Jul 21;14:714. doi: 10.12688/f1000research.165543.1
New Horizons for CCP
1. CCP installs BBSRC ALERT funded Orbitrap Astral - November 2025
2. CCP awarded Gold LEAF accreditation February 2026
