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Immunoextraction of zinc proteins from human plasma using chicken yolk antibodies immobilized onto paramagnetic particles and their electrophoretic analysis

Authors

  • Sona Krizkova,

    1. Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic
    2. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
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  • Marketa Ryvolova,

    1. Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic
    2. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
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  • David Hynek,

    1. Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic
    2. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
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  • Tomas Eckschlager,

    1. Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, University Hospital Motol, Charles University, Prague, Czech Republic
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  • Petr Hodek,

    1. Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic
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  • Michal Masarik,

    1. Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
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  • Vojtech Adam,

    1. Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic
    2. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
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  • Rene Kizek

    Corresponding author
    1. Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
    • Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic
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  • Colour Online: See the article online to view Figs. 1-4 in colour.

Correspondence: Assoc. Prof. Ing. Rene Kizek, Ph.D., Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic

E-mail:kizek@sci.muni.cz

Fax: +420–5-4521–2044

Abstract

Zinc(II) as the only transition metal lacking redox activity is an essential part of approximately 10% proteins as a cofactor of these proteins. Considering the fact that there are numerous zinc(II) containing proteins, proteomics and metallomics studies aimed on them require accurate methods for preparation of real biological samples prior to their subsequent analysis using 2DE and MS. For this purpose, we suggested a new method based on chicken anti-zinc antibodies and magnetizable particles. Antibodies were covalently immobilized to the surface of paramagnetic beads activated with tosyl group. Binding of the antibody to the beads was confirmed by secondary anti-chicken antibody conjugated with horseradish peroxidase. The immunoextraction conditions, such as concentration of the beads (6–18 μg/mL of the sample), time of immunoextraction (6–34 min), pH and composition of the elution buffer, and time of extraction (48–300 s) were optimized. Subsequently, zinc proteins were extracted from human plasma and total concentration of zinc was monitored by electrochemical detection in the extracts. Under optimal conditions it was possible to monitor the proteins and zinc removal from the sample by chip CE, SDS-PAGE, and indirectly using electrochemistry.

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