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Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

Kris Gevaert UGent, Bart Ghesquière UGent, An Staes UGent, Lennart Martens UGent, Jozef Van Damme UGent, Grégoire Thomas and Joël Vandekerckhove (2004) PROTEOMICS. 4(4). p.897-908
abstract
We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
mass spectrometry, cysteine-peptides, non-gel proteomics, MASS-SPECTROMETRY, QUANTITATIVE-ANALYSIS, SERUM PROTEOME, IDENTIFICATION, PROTEINS, QUANTIFICATION, PLASMA, ELECTROPHORESIS, STRATEGY, MIXTURES
journal title
PROTEOMICS
Proteomics
volume
4
issue
4
pages
897 - 908
Web of Science type
Article
Web of Science id
000220763900001
JCR category
BIOCHEMICAL RESEARCH METHODS
JCR impact factor
5.483 (2004)
JCR rank
5/50 (2004)
JCR quartile
1 (2004)
ISSN
1615-9853
DOI
10.1002/pmic.200300641
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
215230
handle
http://hdl.handle.net/1854/LU-215230
date created
2004-05-10 16:28:00
date last changed
2016-12-19 15:38:00
@article{215230,
  abstract     = {We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.},
  author       = {Gevaert, Kris and Ghesqui{\`e}re, Bart and Staes, An and Martens, Lennart and Van Damme, Jozef and Thomas, Gr{\'e}goire and Vandekerckhove, Jo{\"e}l},
  issn         = {1615-9853},
  journal      = {PROTEOMICS},
  keyword      = {mass spectrometry,cysteine-peptides,non-gel proteomics,MASS-SPECTROMETRY,QUANTITATIVE-ANALYSIS,SERUM PROTEOME,IDENTIFICATION,PROTEINS,QUANTIFICATION,PLASMA,ELECTROPHORESIS,STRATEGY,MIXTURES},
  language     = {eng},
  number       = {4},
  pages        = {897--908},
  title        = {Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies},
  url          = {http://dx.doi.org/10.1002/pmic.200300641},
  volume       = {4},
  year         = {2004},
}

Chicago
Gevaert, Kris, Bart Ghesquière, An Staes, Lennart Martens, Jozef Van Damme, Grégoire R Thomas, and Joël Vandekerckhove. 2004. “Reversible Labeling of Cysteine-containing Peptides Allows Their Specific Chromatographic Isolation for Non-gel Proteome Studies.” Proteomics 4 (4): 897–908.
APA
Gevaert, K., Ghesquière, B., Staes, A., Martens, L., Van Damme, J., Thomas, G. R., & Vandekerckhove, J. (2004). Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies. PROTEOMICS, 4(4), 897–908.
Vancouver
1.
Gevaert K, Ghesquière B, Staes A, Martens L, Van Damme J, Thomas GR, et al. Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies. PROTEOMICS. 2004;4(4):897–908.
MLA
Gevaert, Kris, Bart Ghesquière, An Staes, et al. “Reversible Labeling of Cysteine-containing Peptides Allows Their Specific Chromatographic Isolation for Non-gel Proteome Studies.” PROTEOMICS 4.4 (2004): 897–908. Print.