Selenoprotein P analysis in Human Plasma: A Discrepancy Between HPLC Fractionation of Human Plasma with Heparin-Affinity Chromatography and SDS-PAGE with Immunoblot Analysis
Jacobson, GA and Featherstone, AM and Townsend, AT and Lord, R and Peterson, GM (2005) Selenoprotein P analysis in Human Plasma: A Discrepancy Between HPLC Fractionation of Human Plasma with Heparin-Affinity Chromatography and SDS-PAGE with Immunoblot Analysis. Biological Trace Element Research, 107 (3). pp. 213-220.
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Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma–mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.
|Keywords:||Selenoprotein P; selenium; ICP-MS; heparin; SDS-PAGE|
|Deposited By:||Dr Ashley Townsend|
|Deposited On:||06 Jul 2008 19:58|
|Last Modified:||18 Jul 2008 21:07|
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