Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics.
(2019) Nature Communications — Vol. 10, n° 1, p. 331 [1-15] (2019)
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Geladaki, Aikaterini 
Smith, Tom S Lilley, Kathryn S
- Abstract
- The study of protein localisation has greatly benefited from high-throughput methods utilising cellular fractionation and proteomic profiling. Hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) is a well-established method in this area. It achieves high-resolution separation of organelles and subcellular compartments but is relatively time- and resource-intensive. As a simpler alternative, we here develop Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) and compare this method to the density gradient-based hyperLOPIT approach. We confirm that high-resolution maps can be obtained using differential centrifugation down to the suborganellar and protein complex level. HyperLOPIT and LOPIT-DC yield highly similar results, facilitating the identification of isoform-specific localisations and high-confidence localisation assignment for proteins in suborganellar structures, protein complexes and signalling pathways. By combining both approaches, we present a comprehensive high-resolution dataset of human protein localisations and deliver a flexible set of protocols for subcellular proteomics.
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Geladaki, A., Kočevar Britovšek, N., Breckels, L. M., Smith, T. S., Vennard, O. L., Mulvey, C. M., Crook, O. M., Gatto, L., & Lilley, K. S. (2019). Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics. Nature Communications, 10(1), 331 [1-15]. https://doi.org/10.1038/s41467-018-08191-w (Original work published 2019)