Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis |
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Authors: | Daniel J Kutscher Mattias B Fricker Bodo Hattendorf Jörg Bettmer Detlef Günther |
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Institution: | 1.Department for Physical and Analytical Chemistry,University of Oviedo,Oviedo,Spain;2.Department of Chemistry and Applied Biosciences,ETH Zurich,Zurich,Switzerland |
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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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