Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis.
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.
Malmström
Johan
J
Institute for Molecular Systems Molecular Biology, Swiss Federal Institute for Technology Zürich (ETH), Hoenggerberg 8093 Zürich, Switzerland.
Lee
Hookeun
H
Nesvizhskii
Alexey I
AI
Shteynberg
David
D
Mohanty
Sonali
S
Brunner
Erich
E
Ye
Mingliang
M
Weber
Gerhard
G
Eckerskorn
Christoph
C
Aebersold
Ruedi
R
D000818:Animals; D002853:Chromatography, Liquid; D004331:Drosophila melanogaster; D004586:Electrophoresis / Q000379:methods*; D008353:Mannitol; D013058:Mass Spectrometry; D010455:Peptides / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D014508:Urea
D010455:Peptides; D008353:Mannitol; D014508:Urea