Efficient site-specific labeling of proteins via cysteines

Bioconjug Chem. 2008 Mar;19(3):786-91. doi: 10.1021/bc7002499. Epub 2008 Feb 15.


Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ammonium Sulfate / chemistry
  • Coloring Agents
  • Cysteine / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / drug effects
  • Escherichia coli / genetics
  • Fluorescent Dyes
  • Histocompatibility Antigens Class I / chemistry
  • Histocompatibility Antigens Class I / genetics
  • Humans
  • Indicators and Reagents
  • Minor Histocompatibility Antigens
  • Models, Molecular
  • Peptide Initiation Factors / genetics
  • Plasmids / chemistry
  • Plasmids / genetics
  • Proteins / chemistry*
  • Shewanella / chemistry
  • Spectrophotometry, Ultraviolet


  • Coloring Agents
  • Fluorescent Dyes
  • Histocompatibility Antigens Class I
  • Indicators and Reagents
  • MR1 protein, human
  • Minor Histocompatibility Antigens
  • Peptide Initiation Factors
  • Proteins
  • Cysteine
  • Ammonium Sulfate