Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics

• DOI: 10.1038/s41467-018-08191-w
• Publication Date: 2019-01-18T11:06:06Z
• Authors: Aikaterini Geladaki; Nina Kočevar Brit...; Lisa M. Breckels; Tom S. Smith; Owen L. Vennard; Claire M. Mulvey; Oliver M. Crook; Laurent Gatto; Kathryn S. Lilley
• Keywords: Proteomics; 0301 basic medicine; 0303 health sciences; Spatial Analysis; Proteome; Science; Q; Cell Fractionation; Article; Mass Spectrometry; 03 medical and health sciences; Cell Line, Tumor; Centrifugation, Density Gradient; Humans; Ultracentrifugation

AbstractThe 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.