[Sec-to-Cys]selenoprotein - a novel type of recombinant, full-length selenoprotein standard for quantitative proteomics

In this work, we present the first methodical approach to the preparation, molecular characterization and use of a novel type of full-length selenoprotein standard as exemplified for the human plasma selenoproteins, glutathione peroxidase 3 (GPx3) and selenoprotein P (SEPP1), both in non-labeled and stable isotope-labeled forms. Their production was achieved by a cell-free protein synthesis in E. coli extracts. Plasmids with modified coding sequences for GPx3 and SEPP1 were used as DNA templates. In the plasmids, all selenocysteine coding nucleotide triplets (TGA), which in standard conditions serve as stop codons, were site-mutated to cysteine codons (TGT) resulting in exchange of all selenocysteine residues for cysteine residues in the protein amino acid sequence. We named this type of recombinant selenoprotein analog as [Sec-to-Cys]selenoprotein standard. To allow their accurate quantification by inductively coupled plasma mass spectrometry (ICP MS), selenium is introduced into [Sec-to-Cys]selenoprotein standards by replacing Met by SeMet during cell-free protein synthesis. The introduction of e.g. 76 SeMet allows the quantification of [Sec-to-Cys]selenoprotein standards by isotope dilution inductively coupled plasma mass spectrometry (ID ICPMS). Molecular characterization of [Sec-to-Cys]selenoprotein standards is achieved by electrospray tandem mass spectrometry (ESI MS/MS) analysis including a trypsin digestion step. A [Sec-to-Cys]SEPP1 standard containing 76 Se-enriched SeMet was successfully applied as a pseudo-species-specific SEPP1 spike for the quantification of natural SEPP1 in human serum/plasma reference materials (BCR-637 and SRM 1950) by species-specific isotope dilution ICP MS using double affinity high performance liquid chromatography separation. [Sec-to-Cys]selenoprotein standards will help to establish novel, validated and traceable analytical methods for the quantification of human plasma selenoproteins. In this work, we present the first methodical approach to the preparation, molecular characterization and use of a novel type of full-length human selenoprotein standard.