Saturation fluorescence labeling of proteins for proteomic analyses

Elizabeth Pretzer, John E. Wiktorowicz

    Research output: Contribution to journalArticle

    30 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)250-262
    Number of pages13
    JournalAnalytical Biochemistry
    Volume374
    Issue number2
    DOIs
    StatePublished - Mar 15 2008

    Fingerprint

    Proteomics
    Labeling
    Fluorescence
    Coloring Agents
    Thiourea
    Proteins
    Reducing Agents
    Sulfhydryl Compounds
    Carbonic Anhydrase I
    Lactalbumin
    Carbonic Anhydrases
    Phosphopyruvate Hydratase
    Myoglobin
    Pixels
    Disulfides
    Horses
    Costs and Cost Analysis
    Gages
    Imaging techniques
    tris(2-carboxyethyl)phosphine

    Keywords

    • Cys labeling
    • Proteomics
    • Saturation fluorescence labeling
    • Two-dimensional electrophoresis

    ASJC Scopus subject areas

    • Biochemistry
    • Biophysics
    • Molecular Biology

    Cite this

    Saturation fluorescence labeling of proteins for proteomic analyses. / Pretzer, Elizabeth; Wiktorowicz, John E.

    In: Analytical Biochemistry, Vol. 374, No. 2, 15.03.2008, p. 250-262.

    Research output: Contribution to journalArticle

    Pretzer, Elizabeth ; Wiktorowicz, John E. / Saturation fluorescence labeling of proteins for proteomic analyses. In: Analytical Biochemistry. 2008 ; Vol. 374, No. 2. pp. 250-262.
    @article{79bf8f4ce12f482fb44950aea8f28682,
    title = "Saturation fluorescence labeling of proteins for proteomic analyses",
    abstract = "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.",
    keywords = "Cys labeling, Proteomics, Saturation fluorescence labeling, Two-dimensional electrophoresis",
    author = "Elizabeth Pretzer and Wiktorowicz, {John E.}",
    year = "2008",
    month = "3",
    day = "15",
    doi = "10.1016/j.ab.2007.12.014",
    language = "English (US)",
    volume = "374",
    pages = "250--262",
    journal = "Analytical Biochemistry",
    issn = "0003-2697",
    publisher = "Academic Press Inc.",
    number = "2",

    }

    TY - JOUR

    T1 - Saturation fluorescence labeling of proteins for proteomic analyses

    AU - Pretzer, Elizabeth

    AU - Wiktorowicz, John E.

    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

    UR - http://www.scopus.com/inward/record.url?scp=39149130322&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=39149130322&partnerID=8YFLogxK

    U2 - 10.1016/j.ab.2007.12.014

    DO - 10.1016/j.ab.2007.12.014

    M3 - Article

    C2 - 18191033

    AN - SCOPUS:39149130322

    VL - 374

    SP - 250

    EP - 262

    JO - Analytical Biochemistry

    JF - Analytical Biochemistry

    SN - 0003-2697

    IS - 2

    ER -