Hyperplexing: a method for higher-order multiplexed quantitative proteomics provides a map of the dynamic response to rapamycin in yeast

Sci Signal. 2012 Mar 27;5(217):rs2. doi: 10.1126/scisignal.2002548.

Abstract

Large-scale quantitative proteomics can provide a near-global view of cellular protein abundance. Yet, the time, effort, and expertise required to achieve reasonable protein coverage and reliable quantification have limited the broad application of this technology. To fully leverage mass spectrometry for the elucidation of biological systems requires sufficient throughput to monitor dynamic changes across conditions and to enable replicate analysis to provide statistical power. We report a straightforward approach to increase the multiplexing capacity of quantitative mass spectrometry, which provides a platform for the analysis of cellular signaling pathways. Using triplex metabolic labeling and six-plex isobaric tags, we monitored changes in protein abundance from 18 samples simultaneously, performing biological triplicates of a six-point time course of rapamycin-stimulated yeast. The data set provides temporal abundance profiles for thousands of yeast proteins, highlighting the complex cellular roles of the TOR (target of rapamycin) pathway.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / chemistry
  • Carbon Isotopes / chemistry
  • Cations
  • Chromatography, Ion Exchange
  • Immunoblotting
  • Immunosuppressive Agents / pharmacology
  • Isotope Labeling / methods
  • Mass Spectrometry / methods
  • Nitrogen Isotopes / chemistry
  • Proteome / analysis*
  • Proteome / chemistry
  • Proteome / metabolism
  • Proteomics / methods*
  • Reproducibility of Results
  • Saccharomyces cerevisiae / drug effects*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sirolimus / pharmacology*

Substances

  • Amino Acids
  • Carbon Isotopes
  • Cations
  • Immunosuppressive Agents
  • Nitrogen Isotopes
  • Proteome
  • Saccharomyces cerevisiae Proteins
  • Sirolimus