thiourea and tris(2-carboxyethyl)phosphine

We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations, including the disulfide reducing agent tris(2-carboxyethyl)phosphine (TCEP), pH, incubation time, linearity of labeling, and saturating dye/protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific versus nonspecific labeling in the presence of thiourea are also discussed. We found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, and alpha-lactalbumin) is achieved at 50- to 100-fold excess of the uncharged maleimide-functionalized BODIPY dyes over Cys. We confirmed our previous findings, and those of others, that the maleimide dyes are not affected by the presence of 2 M thiourea. Moreover, we established that 2 mM TCEP used as reductant is optimal. We also established that labeling is optimal at pH 7.5 and complete after 30 min. Low nonspecific labeling was gauged by the inclusion of non-Cys-containing proteins (horse myoglobin and bovine carbonic anhydrase) to the labeling mixture. We also showed that the dye exhibits little to no effect on the two-dimensional mobilities of labeled proteins derived from cells.

Topics: Animals; Cattle; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Iodoacetamide; Linear Models; Maleimides; Phosphines; Proteins; Proteomics; Sensitivity and Specificity; Solubility; Staining and Labeling; Sulfhydryl Compounds; Thiourea; Time Factors