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Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
Published on: January 5, 2017
Fractionation of selenium-containing proteins in serum by multiaffinity liquid chromatography before size-exclusion
Oscar Palacios1, Jorge Ruiz Encinar, Dirk Schaumlöffel
1Equipe de Chimie Analytique Bio-inorganique, CNRS UMR 5034, Hélioparc, 2, av. Pr. Angot, 64053, Pau, France.
Abstract:
Immunoaffinity chromatography has been investigated for fractionation of serum into selenoalbumin and true selenoproteins. Among several albumin-depletion kits tested, a multiaffinity column specifically binding albumin and five other major serum proteins provided the best results. It extracted ca 95% of both albumin and selenoalbumin, which enabled interference-free determination of glutathione peroxidase, selenoprotein P, and selenoalbumin by size-exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICPMS). The efficiency of the multiaffinity column did not vary over a period of 18 months. The purity of fractions separated by immunoaffinity LC was confirmed by elution-volume matching with standards in SEC-ICPMS and by selenopeptide mapping in capillary HPLC-ICPMS. Quantification of the selenium distribution among the different proteins in human serum from a control group and from a person on a selenium-rich diet revealed that 67% of the supplemented selenium was incorporated into albumin, 30% into glutathione peroxidase, and 3% into selenoprotein P.
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