Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics.
IF: 17.694
Cited by: 126


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.


Spatial Proteomics

MeSH terms

Cell Fractionation
Cell Line, Tumor
Centrifugation, Density Gradient
Mass Spectrometry
Spatial Analysis


Geladaki, Aikaterini
Kočevar Britovšek, Nina
Breckels, Lisa M
Smith, Tom S
Vennard, Owen L
Mulvey, Claire M
Crook, Oliver M
Gatto, Laurent
Lilley, Kathryn S

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