Minimal, Encapsulated Proteomic-Sample Processing Applied to Copy-Number Estimation in Eukaryotic Cells

Nat Methods. 2014 Mar;11(3):319-24. doi: 10.1038/nmeth.2834. Epub 2014 Feb 2.

Abstract

Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R(2) = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R(2) = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • DNA Contamination
  • Eukaryotic Cells / metabolism*
  • Gene Dosage / genetics*
  • HeLa Cells
  • Humans
  • Proteomics / methods*
  • Reproducibility of Results
  • Saccharomyces cerevisiae Proteins / genetics

Substances

  • Saccharomyces cerevisiae Proteins