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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 (UGent)
(2004) PROTEOMICS. 4(4). p.897-908
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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.
Keywords
mass spectrometry, cysteine-peptides, non-gel proteomics, MASS-SPECTROMETRY, QUANTITATIVE-ANALYSIS, SERUM PROTEOME, IDENTIFICATION, PROTEINS, QUANTIFICATION, PLASMA, ELECTROPHORESIS, STRATEGY, MIXTURES

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Chicago
Gevaert, Kris, Bart Ghesquière, An Staes, Lennart Martens, Jozef Van Damme, Grégoire 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., & 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 G, 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.
@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},
}

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