Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

doi:10.1007/978-1-4939-9736-7_10

. 2019:2049:165-190.

doi: 10.1007/978-1-4939-9736-7_10.

Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

Daniel J H Nightingale^{1

2}, Stephen G Oliver², Kathryn S Lilley^{3

4}

Affiliations

¹ Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
² Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Cambridge, UK.
³ Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. k.s.lilley@bioc.cam.ac.uk.
⁴ Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Cambridge, UK. k.s.lilley@bioc.cam.ac.uk.

PMID: 31602611
DOI: 10.1007/978-1-4939-9736-7_10

Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

Daniel J H Nightingale et al. Methods Mol Biol. 2019.

. 2019:2049:165-190.

doi: 10.1007/978-1-4939-9736-7_10.

Authors

Daniel J H Nightingale^{1

2}, Stephen G Oliver², Kathryn S Lilley^{3

4}

Affiliations

¹ Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
² Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Cambridge, UK.
³ Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. k.s.lilley@bioc.cam.ac.uk.
⁴ Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Cambridge, UK. k.s.lilley@bioc.cam.ac.uk.

PMID: 31602611
DOI: 10.1007/978-1-4939-9736-7_10

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.

Keywords: Organelle; Protein localization; Saccharomyces cerevisiae; Spatial proteomics; Subcellular fractionation; hyperLOPIT.

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References

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1. Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793:650–663 - PubMed - DOI - PMC
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1. Chen XJ, Wang X, Kaufman BA et al (2005) Aconitase couples metabolic regulation to mitochondrial DNA maintenance. Science 80(307):714–717 - DOI

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[1] Delic M, Valli M, Graf AB et al (2013) The secretory pathway: exploring yeast diversity. FEMS Microbiol Rev 37:872–914 - PubMed - DOI - PMC

[2] Delic M, Valli M, Graf AB et al (2013) The secretory pathway: exploring yeast diversity. FEMS Microbiol Rev 37:872–914 - PubMed - DOI - PMC

[3] Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793:650–663 - PubMed - DOI - PMC

[4] Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793:650–663 - PubMed - DOI - PMC

[5] Bardwell L (2005) A walk-through of the yeast mating pheromone response pathway. Peptides 26:339–350 - PubMed - PMC - DOI

[6] Bardwell L (2005) A walk-through of the yeast mating pheromone response pathway. Peptides 26:339–350 - PubMed - PMC - DOI

[7] Gancedo C, Flores C-L, Gancedo JM (2016) The expanding landscape of moonlighting proteins in yeasts. Microbiol Mol Biol Rev 80:765–777 - PubMed - PMC - DOI

[8] Gancedo C, Flores C-L, Gancedo JM (2016) The expanding landscape of moonlighting proteins in yeasts. Microbiol Mol Biol Rev 80:765–777 - PubMed - PMC - DOI

[9] Chen XJ, Wang X, Kaufman BA et al (2005) Aconitase couples metabolic regulation to mitochondrial DNA maintenance. Science 80(307):714–717 - DOI

[10] Chen XJ, Wang X, Kaufman BA et al (2005) Aconitase couples metabolic regulation to mitochondrial DNA maintenance. Science 80(307):714–717 - DOI

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Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

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Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

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