Characterization and Quantification of Selenoprotein P: Challenges to Mass Spectrometry

Abstract

Selenoprotein P (SELENOP) is an emerging marker of the nutritional status of selenium and of various diseases, however, its chemical characteristics still need to be investigated and methods for its accurate quantitation improved. SELENOP is unique among selenoproteins, as it contains multiple genetically encoded SeCys residues, whereas all the other characterized selenoproteins contain just one. SELENOP occurs in the form of multiple isoforms, truncated species and post-translationally modified variants which are relatively poorly characterized. The accurate quantification of SELENOP is contingent on the availability of specific primary standards and reference methods. Before recombinant SELENOP becomes available to be used as a primary standard, careful investigation of the characteristics of the SELENOP measured by electrospray MS and strict control of the recoveries at the various steps of the analytical procedures are strongly recommended. This review critically discusses the state-of-the-art of analytical approaches to the characterization and quantification of SELENOP. While immunoassays remain the standard for the determination of human and animal health status, because of their speed and simplicity, mass spectrometry techniques offer many attractive and complementary features that are highlighted and critically evaluated.

Keywords: biomarker; cancer; mass spectrometry; metrology; selenium; selenocysteine; selenoprotein P.

Figure 1

Putative number of SeCys residues in SELENOP in different organisms (according to [11,12,14]).

Figure 2

Sequence of human SELENOP. (a) Amino-acid sequence of human SELENOP [53]; (b) schematic representation of human SELENOP (on the basis of [47,53] and Uniprot database).

Figure 3

Schematic overview of the principle of HPLC configuration coupled with ICP-MS for SELENOP determination (a) schematic chromatogram of affinity column heparin coupled with ICP-MS (Se-specific detection) [58,64,68], (b) schematic chromatogram of a size exclusion column followed by an affinity column Heparin coupled with ICP-MS (Se-specific detection) [83,99], (c) schematic chromatogram of affinity column heparin followed by a size exclusion column coupled with ICP-MS (Se-specific detection) [77], (d) schematic chromatogram of multi-affinity columns heparin followed by blue-sepharose coupled with ICP-MS (Se-specific detection) [77,78,84,100,101,102,103], (e) schematic chromatogram of multi-affinity columns heparin followed by blue-sepharose and a size exclusion column coupled with ICP-MS (Se-specific detection) [104], (f) schematic chromatogram of a size exclusion column followed by multi-affinity columns heparin and blue-sepharose coupled with ICP-MS (Se-specific detection) [105,106,107], (g) schematic chromatogram of multi-affinity removal column followed by size exclusion column coupled with ICP-MS (Se-specific detection) [108], (h) schematic chromatogram of a strong anion-exchange column coupled with ICP-MS (Se-specific detection) [109,110,111], (Hep: HiTrap Heparin affinity column, SEC: Size-exclusion chromatography, Blue: HiTrap Blue affinity column, MARC: Multi-affinity removal column, SAX: Strong anion-exchange column).

Figure 4

Quantification of SELENOP by laser ablation ICP MS. (a) SDS -PAGE of immunoprecipitated SELENOP from serum. Left lane: albumin standard and antibodies bands; Right lane: sample lane; graph: ⁸⁰Se intensity as a function of position in the gel [65]; (b) Construction of a calibration curve in SDS PAGE—laser ablation ICP MS with GPx [121]. The calibration curve is a linear function of the quantity of protein in the gel. G. Ballihaut, L.E. Kilpatrick, E.L. Kilpatrick, W.C. Davis, Multiple forms of selenoprotein P in candidate human plasma standard reference material, Mettalomics, 2012, 4, 6, 533-538, with permission of Oxford University Press; Reprinted from TrAC Trend in Analytical Chemistry, 26, 3, 2007, G. Ballihaut, C. Pécheyran, S. Monicou, H. Preud’homme, R. Grimaud, R. Lobinski, G. Ballihaut, R. Grimaud, R. Lobinski, Multimode detection (LA-ICP-MS, MALDI-MS and nanoHPLC-ESI-MS²) in 1D and 2D gel-electrophoresis for selenium-containing proteins, 183-190, Copyright (2007), with permission from Elsevier.