Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring.

Research output: Contribution to journalArticle

  • External authors:
  • Craig Lawless
  • Stephen W Holman
  • Philip Brownridge
  • Karin Lanthaler
  • Victoria M Harman
  • Rachel Watkins
  • Dean E Hammond
  • Rebecca L Miller
  • Paul F G Sims
  • Claire E Eyers
  • Robert J Beynon

Abstract

Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference dataset quantifying over 1800 S. cerevisiae proteins by direct means using protein-specific stable-isotope labelled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The SRM-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ~100 million molecules-per-cell, a median of ~1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a 'gold-standard' reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies.

Bibliographical metadata

Original languageEnglish
Pages (from-to)1309-1322
JournalMolecular and Cellular Proteomics
Volume15
Issue number4
DOIs
StatePublished - 10 Jan 2016