Thiol-reactive dyes for fluorescence labeling of proteomic samples

Kamala Tyagarajan, Elizabeth Pretzer, John E. Wiktorowicz

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY FI C1-IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on p/'s of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (Thio-Glo I and Rhodamine Red C2 maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY FI C1-IA or Rhodamine Red C2 maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification.

Original languageEnglish (US)
Pages (from-to)2348-2358
Number of pages11
JournalElectrophoresis
Volume24
Issue number14
DOIs
StatePublished - Jul 2003
Externally publishedYes

Fingerprint

Sulfhydryl Compounds
Proteomics
Labeling
Coloring Agents
Fluorescence
Iodoacetamide
Cadaverine
Thiourea
Rhodamines
Proteins
Peptide Mapping
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
maleimide
Fluorescent Dyes
Ionization
Mass spectrometry
Cysteine
Digestion
Desorption
Buffers

Keywords

  • Cysteine labeling
  • Fluorescence
  • Proteomics
  • Thiourea
  • Tris(2-carboxyethyl) phosphine

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Tyagarajan, K., Pretzer, E., & Wiktorowicz, J. E. (2003). Thiol-reactive dyes for fluorescence labeling of proteomic samples. Electrophoresis, 24(14), 2348-2358. https://doi.org/10.1002/elps.200305478

Thiol-reactive dyes for fluorescence labeling of proteomic samples. / Tyagarajan, Kamala; Pretzer, Elizabeth; Wiktorowicz, John E.

In: Electrophoresis, Vol. 24, No. 14, 07.2003, p. 2348-2358.

Research output: Contribution to journalArticle

Tyagarajan, K, Pretzer, E & Wiktorowicz, JE 2003, 'Thiol-reactive dyes for fluorescence labeling of proteomic samples', Electrophoresis, vol. 24, no. 14, pp. 2348-2358. https://doi.org/10.1002/elps.200305478
Tyagarajan, Kamala ; Pretzer, Elizabeth ; Wiktorowicz, John E. / Thiol-reactive dyes for fluorescence labeling of proteomic samples. In: Electrophoresis. 2003 ; Vol. 24, No. 14. pp. 2348-2358.
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