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

Malmström, Johan LU ; Lee, Hookeun ; Nesvizhskii, Alexey I ; Shteynberg, David ; Mohanty, Sonali ; Brunner, Erich ; Ye, Mingliang ; Weber, Gerhard ; Eckerskorn, Christoph and Aebersold, Ruedi (2006) In Journal of Proteome Research 5(9). p.9-2241
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.... (More)

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.

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publishing date
type
Contribution to journal
publication status
published
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
in
Journal of Proteome Research
volume
5
issue
9
pages
9 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:33748320788
  • pmid:16944936
ISSN
1535-3893
DOI
10.1021/pr0600632
language
English
LU publication?
no
id
3dfcd127-2e6b-4e17-bfda-8f5cafa6fb4b
date added to LUP
2016-11-16 20:38:06
date last changed
2021-04-13 03:16:21
@article{3dfcd127-2e6b-4e17-bfda-8f5cafa6fb4b,
  abstract     = {<p>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.</p>},
  author       = {Malmström, Johan and Lee, Hookeun and Nesvizhskii, Alexey I and Shteynberg, David and Mohanty, Sonali and Brunner, Erich and Ye, Mingliang and Weber, Gerhard and Eckerskorn, Christoph and Aebersold, Ruedi},
  issn         = {1535-3893},
  language     = {eng},
  number       = {9},
  pages        = {9--2241},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Journal of Proteome Research},
  title        = {Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis},
  url          = {http://dx.doi.org/10.1021/pr0600632},
  doi          = {10.1021/pr0600632},
  volume       = {5},
  year         = {2006},
}