IntroductionSelenium (Se) is an essential micronutrient in animals because it promotes the activities of such selenoenzymes as glutathione peroxidases, iodothyronine 5′-deiodinase, and thioredoxin reductase, by existing in the form of selenocysteine (SeCys) in their active centers.1,2In contrast, Se is not an essential element in plants; rather, it is a serviceable element for growth, similar to sodium and silicon. In spite of its non-essentiality, Se metabolism in plants has been attracting much interest for two reasons. One, is its used in phytoremediation for the removal of contaminating Se. Although Se is a useful material in the semiconductor and electronic industries, it is also an environmental contaminant.3Phytoremediation is a low-cost and environmentally friendly technique to remove Se contaminants. Indeed, the Indian mustard,Brassica juncea, has been extensively studied for Se phytoremediation4–6because of its high Se accumulating ability, fast growth, and high biomass. In this plant, absorbed inorganic Se, such as selenite (Se (iv)) and selenate (Se (vi)), is transformed into less toxic forms,i.e., selenoamino acids, such asSe-methylselenocysteine (MeSeCys) and γ-glutamylmethylselenocysteine (GluMeSeCys). The other reason is its use as a nutritional aid in the form of Se supplements or medicines for cancer prevention and other diseases. As inorganic Se salts exhibit substantial toxicities to animals, less toxic Se compounds from plants are useful for this purpose. Indeed, it has been reported that some selenoamino acid derivatives decrease the incidence of prostate, skin, and mammary cancers7–9and exert cytotoxic effects on cultured tumor cells.10–12However, Se metabolism in plants is not fully revealed, and novel selenopeptides and selenometabolites have been reported with the development of analytical techniques based on mass spectrometry.13,14Selenomethionine (SeMet) is one of the key metabolites in selenized yeasts and plants.15–18In selenized yeast, SeMet biosynthesized from inorganic Se is incorporated into proteins and metabolized to other biomolecules such asSe-adenosylselenomethionine (AdoSeMet) derivatives.19,20As already suggested, the replacement of methionine (Met) with SeMet did not significantly alter protein structure in bacteria and fungi.21,22In addition, we have also reported that SeMet, which could not be discriminated from Met, was incorporated into proteins in anin vitrotranslation system using wheat germ extract (WGE),23also, the fluorescence of green fluorescent protein (GFP) and the activity of JNK stimulatory phosphatase-1 (JSP-1) were not affected by the substitution of Met with SeMet byin vitrotranslation.23Thus, the biosynthesis of SeMet-containing proteins is responsible for the detoxification of Se in plants in addition to the biosynthesis of MeSeCys and GluMeSeCys.24On the other hand, although Met is used not only for protein synthesis but also metabolism into coenzymes, such as methylmethionine (MeMet) andS-adenosylmethionine (AdoMet, SAM), as shown in yeast, the metabolism of SeMet into such coenzymes in higher eukaryotes,i.e., plants and animals, has yet to be revealed.Although the inductively coupled plasma mass spectrometer (ICP-MS) is a superior instrument for elemental speciation owing to its high sensitivity and specificity when used in combination with separation instruments such as a high-performance liquid chromatograph (HPLC), the identification of Se compounds by HPLC-ICP-MS is accomplished by matching the retention times of samples with those of authentic standards.25,26Thus, identification by HPLC-ICP-MS is limited to the situation where authentic Se standards are available. As a complementary method to HPLC-ICP-MS, HPLC coupled with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS/MS) is used to identify unknown selenometabolites. Indeed, novel Se-containing metabolites, such as selenosugars, selenoamino acid derivatives, and AdoSeMet derivatives, have been successfully identified with this method.20,27–31In this study, we intend to reveal the biological transformation of SeMet into SeMet derivatives in a model system of higher eukaryotes,i.e., WGE, by identifying Se-containing molecules with HPLC-ICP-MS and HPLC-ESI-MS/MS. As mentioned above, WGE is a good model system to reveal the metabolism of SeMet. In addition, thein vitrometabolism of SeMet was also evaluated in rabbit reticulocyte lysate (RRL), a mammalianin vitrotranslation system. Moreover, we also intend to depict the entire metabolic pathway of SeMet in higher eukaryotes by assigning Se metabolites on the basis of selenometabolomics.