| The role of metallo-proteins in the body and their speciation is of increasing interest in clinical diagnostics because it is well recognized that their properties, function, and regulation in metabolic processes are highly species dependent. Therefore, their use as health markers for in vitro diagnostic, medical care and nutrition is directly related to their biological role. Selenium-containing proteins are important examples of speciation related health-indicators. In this work, significant progress has been made on the development of analytical methods for the speciation of selenoproteins in serum. A complete two-dimensional chromatographic separation of the different species of selenium-containing proteins of clinical interest including albumin, glutathione peroxidase and selenoprotein P is presented. Metal (Co 2+) chelate affinity high performance liquid chromatography (HPLC) was used to extract the proteins from serum, replacing the traditional low-pressure chromatography. Reversed phase chromatography was used for desalting and further separating selenoprotein fractions. The selenoproteins were identified by a combination of UV detection at 280 nm, selenium measurement by DRC-ICP-MS, and peptide mapping and molecular mass determination by MALDI-MS. Quantification of selenium in the selenium-containing protein species was accomplished using HPLC-UV-DRC-ICP-MS analysis employing 82Se stable isotope dilution. The sample was partially digested on line after separation, with nitric acid, using a novel in house designed interface (IRIS Digester-Evaporator, for which a patent has been applied). This interface reduces nitric acid and organic solvent from the liquid flow, and permits direct connection to the DRC-ICP-MS sample introduction system. It is a choice for HPLC-ICP-MS application or for faster on-line sample preparation for ICP and FAAS systems. The 2D chromatography, particularly the IMAC was the major source of variability in the overall method. |
