Selenoprotein P analysis in human plasma: a discrepancy between HPLC fractionation of human plasma with heparin-affinity chromatography and SDS-PAGE with immunoblot analysis.
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.
Jacobson
Glenn A
GA
School of Pharmacy, University of Tasmania, Australia.
Featherstone
Alison M
AM
Townsend
Ashley T
AT
Lord
Roger
R
Peterson
Gregory M
GM
D001769:Blood / Q000378:metabolism; D001774:Blood Chemical Analysis / Q000379:methods*; D002845:Chromatography; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D017279:Selenocysteine / Q000737:chemistry; D051149:Selenoprotein P / Q000032:analysis* / Q000097:blood*; D013997:Time Factors; D014131:Trace Elements
D000939:Epitopes; D051149:Selenoprotein P; D014131:Trace Elements; D017279:Selenocysteine; D006493:Heparin