Abstract
The subcellular localization of proteins is a posttranslational modification of paramount importance. The ability to study subcellular and organelle proteomes improves our understanding of cellular homeostasis and cellular dynamics. In this chapter, we describe a protocol for the unbiased and high-throughput study of protein subcellular localization in the yeast Saccharomyces cerevisiae: hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT), which involves biochemical fractionation of Saccharomyces cerevisiae and high resolution mass spectrometry-based protein quantitation using TMT 10-plex isobaric tags. This protocol enables the determination of the subcellular localizations of thousands of proteins in parallel in a single experiment and thereby deep sampling and high-resolution mapping of the spatial proteome.
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Acknowledgments
We gratefully acknowledge funding from the BBSRC (CASE studentship BB/I016147/1 to K.S.L. and S.G.O.). We thank Mohamed Elzek for critical reading of the manuscript and suggestions on layout and content.
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Nightingale, D.J.H., Oliver, S.G., Lilley, K.S. (2019). Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT. In: Oliver, S.G., Castrillo, J.I. (eds) Yeast Systems Biology. Methods in Molecular Biology, vol 2049. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9736-7_10
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DOI: https://doi.org/10.1007/978-1-4939-9736-7_10
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