Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic.
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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