Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis

Research output: Contribution to journalArticle

Abstract

Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow. By the use of a flat pI gradient and a mannitol and urea based separation media we were able to perform high-throughput proteome analysis with improved interfacing between FFE and RPLC-MS/MS. The developed protocol was applied to a cytosolic fraction from Schneider S2 cells from Drosophila melanogaster, resulting in the identification of more than 10,000 unique peptides with high probability. To improve the accuracy of the peptide identification following FFE-IEF we incorporated the pI information as an additional parameter into a statistical model for discrimination between correct and incorrect peptide assignments to MS/MS spectra.

Details

Authors
  • Johan Malmström
  • Hookeun Lee
  • Alexey I Nesvizhskii
  • David Shteynberg
  • Sonali Mohanty
  • Erich Brunner
  • Mingliang Ye
  • Gerhard Weber
  • Christoph Eckerskorn
  • Ruedi Aebersold
External organisations
  • ETH Zürich
Research areas and keywords

Keywords

  • Animals, Chromatography, Liquid, Drosophila melanogaster, Electrophoresis, Mannitol, Mass Spectrometry, Peptides, Proteomics, Urea, Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't
Original languageEnglish
Pages (from-to)2241-9
Number of pages9
JournalJournal of Proteome Research
Volume5
Issue number9
Publication statusPublished - 2006 Sep
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes