TY - JOUR
T1 - Saturation fluorescence labeling of proteins for proteomic analyses
AU - Pretzer, Elizabeth
AU - Wiktorowicz, John E.
N1 - Funding Information:
The authors acknowledge the excellent technical assistance in generating the data for Fig. 7 by Susan Stafford of the Biomolecular Resource Facility at the University of Texas Medical Branch. This work was supported in part by NIH/NHLBI contract N01-HV-28184 to the University of Texas Medical Branch (A. Kurosky, principal investigator) and NHLBI SCCOR P50HL083794-01 to the University of Texas Health Science Center at Houston (D. Milewicz, principal investigator). The authors also acknowledge support from Lynx Therapeutics, where the bulk of this work was performed.
PY - 2008/3/15
Y1 - 2008/3/15
N2 - 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 α-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.
AB - 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 α-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.
KW - Cys labeling
KW - Proteomics
KW - Saturation fluorescence labeling
KW - Two-dimensional electrophoresis
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U2 - 10.1016/j.ab.2007.12.014
DO - 10.1016/j.ab.2007.12.014
M3 - Article
C2 - 18191033
AN - SCOPUS:39149130322
SN - 0003-2697
VL - 374
SP - 250
EP - 262
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -