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 Fl C~1~โIA 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__I__'s of standard proteins. Some iodoacetamide dyes, (5โTMRIA and eosinโ5โiodoacetamide) and some maleimide dyes (ThioGloย Iย and Rhodamine Red C~2~ maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2โcarboxyethyl)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 Fl C~1~โIA or Rhodamine Red C~2~ maleimide was also demonstrated to be compatible with inโgel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionizationโmass spectrometry and allowed successful protein identification.