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Selenoprotein P analysis in Human Plasma: A Discrepancy Between HPLC Fractionation of Human Plasma with Heparin-Affinity Chromatography and SDS-PAGE with Immunoblot Analysis
journal contribution
posted on 2023-05-26, 16:22 authored by Jacobson, GA, Featherstone, AM, Ashley TownsendAshley Townsend, Lord, R, Gregory PetersonGregory PetersonSeveral 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‚Äö-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‚Äö-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.
History
Publication title
Biological Trace Element ResearchVolume
107Article number
3Number
3Pagination
213-220Publication status
- Published
Repository Status
- Restricted
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