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Related Experiment Videos

A Protocol to Map the Spatial Proteome Using HyperLOPIT in Saccharomyces cerevisiae.

Daniel J H Nightingale1,2, Kathryn S Lilley1,2, Stephen G Oliver2

  • 1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, United Kingdom.

Bio-Protocol
|March 3, 2021
PubMed
Summary

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This study presents a detailed protocol for mapping the subcellular localization of thousands of proteins in yeast using hyperplexed Localization of Organelle Proteins by Isotope Tagging (hyperLOPIT). This method provides a high-resolution spatial proteome map for enhanced cellular understanding.

Area of Science:

  • Proteomics
  • Cell Biology
  • Systems Biology

Background:

  • Accurate subcellular protein localization is crucial for understanding cellular functions and biological processes.
  • Existing methods for protein localization analysis have limitations in scale and resolution.
  • Systems-level analysis of the proteome's spatial organization is essential for a comprehensive view of cellular mechanisms.

Purpose of the Study:

  • To provide an optimized protocol for the systems-level study of subcellular localization in Saccharomyces cerevisiae.
  • To enable high-resolution, unbiased, and parallel analysis of thousands of protein localizations within a single experiment.
  • To generate a spatial proteome map for yeast.

Main Methods:

  • Utilizes a tailored version of hyperplexed Localization of Organelle Proteins by Isotope Tagging (hyperLOPIT) optimized for yeast.
Keywords:
Nitrogen cavitationQuantitative proteomicsSaccharomyces cerevisiaeSpatial proteomicsSubcellular fractionationYeast

Related Experiment Videos

  • Protocol includes cell culture, nitrogen cavitation lysis, subcellular fractionation, and Western blot validation.
  • Involves TMT isobaric tagging and mass spectrometric analysis, with downstream data processing for spatial proteome mapping.

    • Main Results:

    • The protocol enables the unbiased, systems-level, and high-resolution analysis of thousands of protein localizations simultaneously.
    • Successfully generates a spatial proteome map of Saccharomyces cerevisiae.
    • Components of the protocol, such as lysis and Western blotting, can be used independently.

    Conclusions:

    • The presented hyperLOPIT protocol is a powerful tool for comprehensive subcellular localization studies in yeast.
    • This method significantly advances the understanding of protein function and cellular organization at a systems level.
    • The protocol facilitates high-throughput and high-resolution mapping of the spatial proteome.