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Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

  • Daniel J. H. Nightingale
  • Stephen G. Oliver
  • Kathryn S. LilleyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2049)

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.

Key words

Saccharomyces cerevisiae hyperLOPIT Organelle Protein localization Subcellular fractionation Spatial proteomics 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Daniel J. H. Nightingale
    • 1
    • 2
  • Stephen G. Oliver
    • 2
  • Kathryn S. Lilley
    • 1
    • 2
    Email author
  1. 1.Cambridge Centre for Proteomics, Department of BiochemistryUniversity of CambridgeCambridgeUK
  2. 2.Cambridge Systems Biology Centre, Department of BiochemistryUniversity of CambridgeCambridgeUK

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