Abstract
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 160 fmol 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%.
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Acknowledgements
This work was supported by the Spanish MICINN (Spanish Ministry for Science and Innovation, Grant No. CTQ2008-01725). Furthermore, D.J.K. acknowledges a Ph.D. grant from MICINN and J.B. a contract within the Ramón y Cajal program of MICINN. The group of Prof. Dr. D. Hilvert, ETH Zurich, is kindly acknowledged for their support in the gel electrophoretic separations.
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Published in the special issue Plasma Spectrochemistry with guest editors Juan Castillo and Martín Resano.
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Kutscher, D.J., Fricker, M.B., Hattendorf, B. et al. Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis. Anal Bioanal Chem 401, 2691–2698 (2011). https://doi.org/10.1007/s00216-011-5199-5
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DOI: https://doi.org/10.1007/s00216-011-5199-5