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Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics
Aikaterini Geladaki1,2, Nina Kočevar Britovšek1, Lisa M Breckels1
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Nature Communications
|January 20, 2019
Summary
A new method, Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC), simplifies organelle protein mapping. LOPIT-DC provides high-resolution human protein localisation data comparable to the hyperLOPIT method.
Area of Science:
- Proteomics and Cell Biology
- Subcellular and Organelle Research
Background:
- High-throughput protein localisation studies are crucial for understanding cellular functions.
- Established methods like hyperLOPIT offer high resolution but are resource-intensive.
Purpose of the Study:
- To develop and validate a simpler, more accessible method for high-resolution protein localisation.
- To compare the novel LOPIT-DC method with the established hyperLOPIT technique.
Main Methods:
- Development of Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC).
- Comparative proteomic profiling of human cell lysates using LOPIT-DC and hyperLOPIT.
Main Results:
- LOPIT-DC successfully generates high-resolution protein localisation maps, including suborganellar and protein complex levels.
- LOPIT-DC results are highly similar to those obtained by the density gradient-based hyperLOPIT method.
- Isoform-specific localisations and high-confidence assignments for proteins in various cellular structures were identified.
Conclusions:
- LOPIT-DC is a viable and simpler alternative to hyperLOPIT for subcellular proteomics.
- Combining LOPIT-DC and hyperLOPIT provides a comprehensive human protein localisation dataset and flexible protocols.
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