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Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis

Kutscher, Daniel ; Fricker, Mattias ; Hattendorf, Bodo ; Bettmer, Jörg ; Günther, Detlef

In: Analytical and Bioanalytical Chemistry, 2011, vol. 401, no. 9, p. 2691-2698

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    Titre
    • Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis
    Auteur
    • Kutscher, Daniel. Department for Physical and Analytical Chemistry, University of Oviedo, C/ Julián Clavería 8, 33006, Oviedo, Spain
    • Fricker, Mattias. Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich, Switzerland
    • Hattendorf, Bodo. Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich, Switzerland
    • Bettmer, Jörg. Department for Physical and Analytical Chemistry, University of Oviedo, C/ Julián Clavería 8, 33006, Oviedo, Spain
    • Günther, Detlef. Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Analytical and Bioanalytical Chemistry, 2011, vol. 401, no. 9, p. 2691-2698. Springer-Verlag
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:314625
    Summary
    A method was developed for the precise and accurate determination of ovalbumin labelled with p-hydroxy-mercuribenzoic acid (pHMB) using polyacrylamide gel electrophoresis with ns-laser ablation-inductively coupled plasma mass spectrometry. Following systematic optimisation of the ablation process in terms of detection sensitivity, two different quantification strategies were applied: external calibration using standards of the derivatized protein after 13C+ normalization and, as a proof of concept, label-specific isotope dilution analysis (IDA) using pHMB enriched in the isotope 199Hg. Due to the inhomogeneous distribution of the protein within the gel bands, it could be demonstrated that the IDA approach was superior in terms of precision and accuracy. Furthermore, it permits a reliable quantification, if more complex separation protocols are applied, as typically occurring analyte loss and degradation can be compensated for as soon as complete mixture of spike and sample is achieved. The estimated limit of detection was 160fmol in the case of ovalbumin. In contrast to earlier studies using metals naturally present in proteins, no loss of mercury was observed during separation under denaturing conditions and other sample preparation steps. Using label-specific IDA, the measured isotope ratios in the gel corresponded to recoveries between 95% and 103%