Selenium Decipher: Trapping of Native Selenomethionine-Containing Peptides in Selenium-Enriched Milk and Unveiling the Deterioration after Ultrahigh-Temperature Treatment.

Selenopeptide identification relies on databases to interpret the selenopeptide spectra. A common database search strategy is to set selenium as a variable modification instead of sulfur on peptides. However, this approach generally detects only a fraction of selenopeptides. An alternative approach, termed Selenium Decipher, is proposed in the present study. It involves identifying collision-induced dissociation-cleavable selenomethionine-containing peptides by iteratively matching the masses of seleno-amino acids in selenopeptide spectra. This approach uses variable-data-independent acquisition (vDIA) for peptide detection, providing a flexible and customizable window for secondary mass spectral fragmentation. The attention mechanism was used to capture global information on peptides and determine selenomethionine-containing peptide backbones. The core structure of selenium on selenomethionine-containing peptides generates a series of fragment ions, namely, C3H7Se+, C4H10NSe+, C5H7OSe+, C5H8NOSe+, and C7H11N2O2Se+, with known mass gaps during higher-energy collisional dissociation (HCD) fragmentation. De-selenium spectra are generated by removing selenium originating from selenium replacement and then reassigning the precursors to peptides. Selenium-enriched milk is obtained by feeding selenium-rich forage fed to cattle, which leads to the formation of native selenium through biotransformation. A novel antihypertensive selenopeptide Thr-Asp-Asp-Ile-SeMet-Cys-Val-Lys TDDI(Se)MCVK was identified from selenium-enriched milk. The selenopeptide (IC50 = 60.71 µM) is bound to four active residues of the angiotensin-converting enzyme (ACE) active pocket (Ala354, Tyr523, His353, and His513) and two active residues of zinc ligand (His387 and Glu411) and exerted a competitive inhibitory effect on the spatial blocking of active sites. The integration of vDIA and the iteratively matched seleno-amino acids was applied for Selenium Decipher, which provides high validity for selenomethionine-containing peptide identification.

82Se Metabolically-Labeled Yeast as a Matrix-Matched Isotope Dilution Standard for Quantification of Selenomethionine.

Selenized yeast is commonly used as a highly bioavailable source of selenium in dietary supplements and feed additives and is used in research settings in various disciplines due to the large number of selenium-containing metabolites formed during growth. With the selenomethionine being the major form of selenium present in selenized yeasts, its accurate quantitation is essential, however, values are frequently underestimated due to the costly and time-consuming hydrolysis-based sample preparation required to release the selenoamino acid from proteins for analysis. The National Research Council Canada has developed an 82-Se-enriched selenized yeast Certified Reference Material, SEEY-1 (DOI: 10.4224/crm.2023.seey-1) intended to be used as a matrix-matched spike material for isotope dilution analysis of selenized yeasts. The total selenium and selenomethionine contents of SEEY-1 were determined to be 322.1 ± 4.8 mg/kg (k = 2) and 635.6 ± 16.8 mg/kg (k = 2), respectively. Here we present results on the preparation of the 82-Se-enriched yeast, the certification process, and provide an example of the use of SEEY-1 as a matrix-matched spike for the analysis of selenomethionine in a sample of selenized yeast. We demonstrate here that SEEY-1 is able to compensate for the partial digestion of yeast proteins and provide reliable analytical data on Se amino acid content in under an hour instead of the 16 hours required for conventional complete acid hydrolysis.

Chalcogen Bonds Involving Selenium in Protein Structures.

Chalcogen bonds are the specific interactions involving group 16 elements as electrophilic sites. The role of chalcogen atoms as sticky sites in biomolecules is underappreciated, and the few available studies have mostly focused on S. Here, we carried out a statistical analysis over 3562 protein structures in the Protein Data Bank (PDB) containing 18 266 selenomethionines and found that Se···O chalcogen bonds are commonplace. These findings may help the future design of functional peptides and contribute to understanding the role of Se in nature.

Charge Assisted S/Se Chalcogen Bonds in SAM Riboswitches: A Combined PDB and ab Initio Study.

In this study, we provide experimental (Protein Data Bank (PDB) inspection) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the involvement of charge assisted chalcogen bonding (ChB) interactions in the recognition and folding mechanisms of S-adenosylmethionine (SAM) riboswitches. Concretely, an initial PDB search revealed several examples where ChBs between S-adenosyl methionine (SAM)/adenosyl selenomethionine (EEM) molecules and uracil (U) bases belonging to RNA take place. While these interactions are usually described as a merely Coulombic attraction between the positively charged S/Se group and RNA, theoretical calculations indicated that the σ holes of S and Se are involved. Moreover, computational models shed light on the strength and directionality properties of the interaction, which was also further characterized from a charge-density perspective using Bader's "Atoms in Molecules" (AIM) theory, Non-Covalent Interaction plot (NCIplot) visual index, and Natural Bonding Orbital (NBO) analyses. As far as our knowledge extends, this is the first time that ChBs in SAM-RNA complexes have been systematically analyzed, and we believe the results might be useful for scientists working in the field of RNA engineering and chemical biology as well as to increase the visibility of the interaction among the biological community.

Analytical Problems in Separation of Selenomethionine and Its Oxidative Product in HILIC HPLC.

Selenomethionine (SeMet) is one of the main selenium forms in foods and supplements. Determining its presence in natural food samples creates difficulties due to possible oxidation processes. The objective of this study was to evaluate the possible degradation of SeMet in water extracts of green teas, one of the most consumed beverages worldwide. Such a medium has not been investigated at this time. The HILIC-HPLC MS/MS method with different stationary phases was used to achieve the satisfactory separation of SeMet and selenomethionine oxide (SeMetO). The addition of dithiothreitol and ß-mercaptoethanol, recommended to ensure that SeMet is kept in the reduced form, was also evaluated. The best separation was achieved using the zwitterionic HILIC stationary phase coupled to mass spectrometry and MeOH with water (85/15, v/v) as the eluent. Extraction was done with hot water with the addition of ß-mercaptoethanol. The infusions prepared from Lung-Ching teas (from the Zhejiang Province in China) contained the highest concentration of selenium in a typical cup of tea (12.5-17.3 µg L-1). For other tested teas it decreased in the following order: Yunnan > Dilmah > Lipton. For Lung-Ching teas, the sum of concentrations of SeMet and SeMetO corresponded to about 46-63% of the total selenium in their extracts.

Selenium Biofortification: Roles, Mechanisms, Responses and Prospects.

The trace element selenium (Se) is a crucial element for many living organisms, including soil microorganisms, plants and animals, including humans. Generally, in Nature Se is taken up in the living cells of microorganisms, plants, animals and humans in several inorganic forms such as selenate, selenite, elemental Se and selenide. These forms are converted to organic forms by biological process, mostly as the two selenoamino acids selenocysteine (SeCys) and selenomethionine (SeMet). The biological systems of plants, animals and humans can fix these amino acids into Se-containing proteins by a modest replacement of methionine with SeMet. While the form SeCys is usually present in the active site of enzymes, which is essential for catalytic activity. Within human cells, organic forms of Se are significant for the accurate functioning of the immune and reproductive systems, the thyroid and the brain, and to enzyme activity within cells. Humans ingest Se through plant and animal foods rich in the element. The concentration of Se in foodstuffs depends on the presence of available forms of Se in soils and its uptake and accumulation by plants and herbivorous animals. Therefore, improving the availability of Se to plants is, therefore, a potential pathway to overcoming human Se deficiencies. Among these prospective pathways, the Se-biofortification of plants has already been established as a pioneering approach for producing Se-enriched agricultural products. To achieve this desirable aim of Se-biofortification, molecular breeding and genetic engineering in combination with novel agronomic and edaphic management approaches should be combined. This current review summarizes the roles, responses, prospects and mechanisms of Se in human nutrition. It also elaborates how biofortification is a plausible approach to resolving Se-deficiency in humans and other animals.

77Se NMR Probes the Protein Environment of Selenomethionine.

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se NMR to the nucleus' chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological 77Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution-state 77Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se NMR experiments that exploit selenomethionine to probe proteins.

Water-Mediated Selenium Hydrogen-Bonding in Proteins: PDB Analysis and Gas-Phase Spectroscopy of Model Complexes.

High-resolution X-ray crystallography and two-dimensional NMR studies demonstrate that water-mediated conventional hydrogen-bonding interactions (N-H···N, O-H···N, etc.) bridging two or more amino acid residues contribute to the stability of proteins and protein-ligand complexes. In this work, we have investigated single water-mediated selenium hydrogen-bonding interactions (unconventional hydrogen-bonding) between amino acid residues in proteins through extensive protein data bank (PDB) analysis coupled with gas-phase spectroscopy and quantum chemical calculation of a model complex consisting of indole, dimethyl selenide, and water. Here, indole and dimethyl selenide represent the amino acid residues tryptophan and selenomethionine, respectively. The current investigation demonstrates that the most stable structure of the model complex observed in the IR spectroscopy mimics single water-mediated selenium hydrogen-bonded structural motifs present in the crystal structures of proteins. The present work establishes that water-mediated Se hydrogen-bonding interactions are ubiquitous in proteins and the number of these interactions observed in the PDB is more than that of direct Se hydrogen-bonds present there.

Isolation and identification of immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates.

The RAW264.7 cell model was employed to screen immunomodulatory selenium-containing peptides from selenium-enriched rice protein hydrolysates (SPHs). Moreover, the selenium-containing peptides of high-activity protein hydrolysates were purified by Sephadex G-25, and identified by reversed phase ultra performance liquid chromatography coupled with triple quadrupole time-of-flight mass spectrometry. The results showed that 25 peptide sequences containing selenomethionine (SeMet) information above 90% of probability confidence were found in a fraction of alcalase hydrolysates. SeMDPGQQ and TSeMMM of 100% probability confidence were speculated as two novel selenium-containing peptide sequences. The artificially synthesized peptide TSeMMM was subsequently verified by an excellent immunomodulatory activity at a concentration of 80 µg/mL. In conclusion, the immunomodulatory activity of SPHs was correlated to SeMet sequence in the structure of selenium-containing peptides, and TSeMMM with a stronger immunomodulatory activity demonstrated potential as functional food additives for improving human health.

Selenomethionine and Methioninase: Selenium Free Radical Anticancer Activity.

Colloidal selenium, was first used to treat cancer as early as 1911 in both humans and mice. Selenium was identified as the toxic component in forage plants of sheep, cattle, and horses in the 1930s. The animal toxicity of selenium compounds was determined to be from the metabolism by animals of the elevated concentrations of Se-methylselenocysteine and selenomethionine in plants. The metabolism of both Se-methylselenocysteine and selenomethionine by animals gives rise to the metabolite, methylselenide (CH3Se-), which if in sufficient concentration oxidizes thiols and generates superoxide and other reactive oxygen species. Cancer cells that may overly express methionine gamma-lyase, or beta-lyase (methioninase), by induced viral genomic expression, are susceptible to free radical-induced apoptosis from selenomethionine or Se-methylselenocysteine supplementation.

Generating cellulose-agar composite hydrogels for uptake-release kinetic studies of selenate and selenomethionine.

Cellulose-agar (CAB) composite hydrogel beads were generated for the uptake-release kinetics studies of Se(VI) and selenomethionine (SeMt) from water medium. The objective of this work is to analyze the surface structure, gel properties, thermal stability and chemical functionalities responsible for the adsorption of Se(VI) and SeMt. We propose here a possible mechanism for the adsorptions. Adsorption isotherms are in good agreement with the Freundlich model, yielding a high adsorption capacity for the CAB composite. Maximum adsorption capacity of Se(VI) and SeMt were found to be 7.083 mg g-1 and 34.639 mg g-1 respectively. The mean free energy of adsorption (E*) value was found to be 0.0423 kJ mol-1 and 0.329 kJ mol-1 of Se(VI) and SeMt respectively. 1 M HCl and 0.1 M HCl were able to desorb Se(VI) and SeMt respectively from CAB. The adsorption of Se(VI) was significantly reduced if As(III), Cr(III) and Hg(II) were present as complementary ions in the medium. Similar studies with pristine cellulose beads (CB) yielded insignificant uptake properties.

Seleno-l-methionine and l-ascorbic acid differentiate the biological activity of doxorubicin and its metal complexes as a new anticancer drugs candidate.

The most important problems of anti-cancer therapy include the toxicity of the drugs applied to healthy cells and the multi-drug cells resistance to chemotherapeutics. One of the most commonly used anticancer drugs is doxorubicin (DOX) used to treat certain leukemias and non-Hodgkin's lymphomas, as well as bladder, breast, stomach, lung, ovarian, thyroid, multiple myeloma and other cancers. Preliminary studies showed that metal complex with DOX improve its cytostatic activity with changes in their molecular structure and distribution of electrons, resulting in a substantial change of its biological activity (including antitumor activity). Thus, there is a chance to receiving derivatives of DOX with low toxicity for the healthy body cells, thus increasing its therapeutic selectivity. In the present study we examined the influence of Mn, Mg, Fe, Co and Ni, seleno-l-methionine and vitamin C on biological activity of DOX in prokaryotic model - Escherichia coli RFM443, with plasmid transcriptional fusion of recA promoter and luxCDABE as a reporter gene. Cytotoxic potency of tested chemicals was calculated on the basis of the bacteria culture growth inhibition (GI%) values. Genotoxic properties were calculated on the basis of the fold increase (FI) of relative luminescence units (RLU) values compared to control. Obtained results showed that doxorubicin metal complexes particularly with Ni, Co and Fe increased the cyto- and genotoxic activities of DOX. Bacteria culture supplemented with SeMet and vitamin C differentiate the DOX and its metal complexes toxicity. It seems, that DOX-Ni, DOX-Fe and DOX-Co complexes could be potent cytostatic drug candidates. Moreover, we noticed different sensitivity of recA::luxCDABE for 3 h and 24 h cultures of bacteria strain. It suggests, that the potency of genetic construct reactivity- recA::luxCDABE in E. coli depends on the growth-phase of bacterial culture.

Quantification of selenomethionine in plasma using UPLC-MS/MS after the oral administration of selenium-enriched yeast to rats.

The in vivo determination of selenomethionine (SeMet) converted from selenium-enriched yeast (SeY) rather than the determination of in vitro hydrolyzed SeMet is a better parameter for the evaluation of SeY quality. A UPLC-MS/MS method was developed for the quantification of SeMet in rat plasma and the oral bioavailability of SeMet converted from SeY in rats. After a simple extraction with perchloric acid, SeMet and the internal standard methylselenocysteine (MSC) were separated on a C18 column with isocratic elution of water:acetonitrile:formic acid (99:1:0.1, v/v/v) and detected in the multiple reaction monitoring mode. The method was accurate (92.6-104.3%) and precise (1.8-11.0%), and the recovery was 79.4-95.4%. It was successfully applied to pharmacokinetic and bioavailability studies of SeY in rats following the intravenous administration of SeMet and intragastric administration of SeY. SeMet in vivo, converted from SeY, is reported for the first time, and the results suggested that the SeY bioavailability in rats is 87%.

Catalytic oxidant scavenging by selenium-containing compounds: Reduction of selenoxides and N-chloramines by thiols and redox enzymes.

Myeloperoxidase produces strong oxidants during the immune response to destroy invading pathogens. However, these oxidants can also cause tissue damage, which contributes to the development of numerous inflammatory diseases. Selenium containing compounds, including selenomethionine (SeMet) and 1,4-anhydro-5-seleno-D-talitol (SeTal), react rapidly with different MPO-derived oxidants to form the respective selenoxides (SeMetO and SeTalO). This study investigates the susceptibility of these selenoxides to undergo reduction back to the parent compounds by intracellular reducing systems, including glutathione (GSH) and the glutathione reductase and thioredoxin reductase systems. GSH is shown to reduce SeMetO and SeTalO, with consequent formation of GSSG with apparent second order rate constants, k2, in the range 103-104M-1s-1. Glutathione reductase reduces both SeMetO and SeTalO at the expense of NADPH via formation of GSSG, whereas thioredoxin reductase acts only on SeMetO. The presence of SeMet and SeTal also increased the rate at which NADPH was consumed by the glutathione reductase system in the presence of N-chloramines. In contrast, the presence of SeMet and SeTal reduced the rate of NADPH consumption by the thioredoxin reductase system after addition of N-chloramines, consistent with the rapid formation of selenoxides, but only slow reduction by thioredoxin reductase. These results support a potential role of seleno compounds to act as catalytic scavengers of MPO-derived oxidants, particularly in the presence of glutathione reductase and NADPH, assuming that sufficient plasma levels of the parent selenoether can be achieved in vivo following supplementation.

Deproteinization assessment using isotopically enriched compounds to trace the coprecipitation of low-molecular-weight selenium species with proteins.

Studies have shown that information related to the presence of low-molecular-weight metabolites is frequently lost after deproteinization of complex matrices, such as blood and plasma, during sample preparation. Therefore, the effect of several deproteinization reagents on low-molecular-weight selenium species has been compared by species-specific isotope labeling. Two isotopically enriched selenium tracers were used to mimic models of small inorganic anionic (77Se-selenite) and organic zwitterionic (76Se-selenomethionine) species. The results presented here show that the use of a methanol-acetonitrile-acetone (1:1:1 v/v/v) mixture provided approximately two times less tracer loss from plasma samples in comparison with the classic procedure using acetonitrile, which may not be optimal as it leads to important losses of low-molecular-weight selenium species. In addition, the possible interactions between selenium tracers and proteins were investigated, revealing that both coprecipitation phenomena and association with proteins were potentially responsible for selenite tracer losses during protein precipitation in blood samples. However, coprecipitation phenomena were found to be fully responsible for losses of both tracers observed in plasma samples and of the selenomethionine tracer in blood samples. This successfully applied strategy is anticipated to be useful for more extensive future studies in selenometabolomics.

Spectroscopic Evidences for Strong Hydrogen Bonds with Selenomethionine in Proteins.

Careful protein structure analysis unravels many unknown and unappreciated noncovalent interactions that control protein structure; one such unrecognized interaction in protein is selenium centered hydrogen bonds (SeCHBs). We report, for the first time, SeCHBs involving the amide proton and selenium of selenomethionine (Mse), i.e., amide-N-H···Se H-bonds discerned in proteins. Using mass selective and conformer specific high resolution vibrational spectroscopy, gold standard quantum chemical calculations at CCSD(T), and in-depth protein structure analysis, we establish that amide-N-H···Se and amide-N-H···Te H-bonds are as strong as conventional amide-NH···O and amide-NH···O═C H-bonds despite smaller electronegativity of selenium and tellurium than oxygen. It is in fact, electronegativity, atomic charge, and polarizability of the H-bond acceptor atoms are at play in deciding the strength of H-bonds. The amide-N-H···Se and amide-N-H···Te H-bonds presented here are not only new additions to the ever expanding world of noncovalent interactions, but also are of central importance to design new force-fields for better biomolecular structure simulations.

Bioaccessibility of selenium, selenomethionine and selenocysteine from foods and influence of heat processing on the same.

Selenium (Se) is an essential nutrient with diverse physiological functions. The selenium content of commonly consumed cereals, pulses and green leafy vegetables (GLV) was determined. Bioaccessibility of Se, and its organic forms selenomethionine (SeMet), and selenocysteine (SeCys2) was also examined, and the effect of heat processing on the same was studied. The bioaccessibility of Se in cereals ranged from 10% to 24%, that of pulses was between 12% and 29%, and of GLV, 10-31%. The concentration of SeMet in the dialysates of the cereals, pulses and GLV ranged from 5.15 to 28.7, 2.7 to 36.2, and 0.03 to 5ngg(-1), respectively. The concentration of SeCys2 in the dialysates of the foods examined was negligible. Heat processing significantly decreased the bioaccessibility of Se, SeMet and SeCys2. This is the first report on the bioaccessibility of Se and its major organic forms from commonly consumed staples, and the effect of heat processing on the same.

An efficient method for the synthesis of selenium modified nucleosides: its application in the synthesis of Se-adenosyl-L-selenomethionine (SeAM).

In this paper, we report that a versatile method for the synthesis of 5'-selenium modified nucleosides has been explored on the basis of a 2-(trimethylsilyl)ethyl (TSE) selenyl group as a selenating donor. We demonstrate the broad utility of this method through direct introduction of various functional groups into 5'-TSE-selenonucleosides. This original method offers additional advantages for the preparation of these compounds, such as high functional group tolerance, ready availability of various electrophilic reagents, mild conditions, simple operation, and good yields. The utility of this approach is further demonstrated by the synthesis of Se-adenosyl-L-selenomethionine (SeAM) as a chemical reporter for methyltransferases.

Multiple crystal forms of N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus.

Native N,N'-diacetylchitobiose deacetylase from Pyrococcus furiosus (Pf-Dac) and its selenomethionine derivative (Se-Pf-Dac) were crystallized and analyzed in the presence and absence of cadmium ion. The four crystal structures fell into three different crystal-packing groups, with the cadmium-free Pf-Dac and Se-Pf-Dac belonging to the same space group, with homologous unit-cell parameters. The crystal structures in the presence of cadmium contained distorted octahedral cadmium complexes coordinated by three chlorides, two O atoms and an S or Se atom from the N-terminal methionine or selenomethionine, respectively. The N-terminal cadmium complex was involved in crystal contacts between symmetry-related molecules through hydrogen bonding to the N-termini. While all six N-termini of Se-Pf-Dac were involved in cadmium-complex formation, only two of the Pf-Dac N-termini participated in complex formation in the Cd-containing crystal, resulting in different crystal forms. These differences are discussed in light of the higher stability of the Cd-Se bond than the Cd-S bond. This work provides an example of the contribution of cadmium towards determining protein crystal quality and packing depending on the use of the native protein or the selenomethionine derivative.

Evaluation of the metabolic chiral inversion of d-selenomethionine in rats by stable isotope dilution gas chromatography-mass spectrometry.

The stereoselective pharmacokinetics of selenomethionine enantiomers in rats has been studied to evaluate the chiral inversion of D-selenomethionine to the L-enantiomer. After bolus intravenous administration of D- or L-selenomethionine to rats, the plasma concentrations of these two enantiomers were determined by stereoselective gas chromatography-mass spectrometry with selected ion monitoring. This method involved derivatization of selenomethionine enantiomers with HCl in methanol to form methyl ester followed by N-acylation with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form the diastereomeric amide, and separation of the diastereomer on GC with an achiral column. Plasma concentrations of administered D- and L-selenomethionine declined with terminal half-lives of 96 ± 17 min and 91 ± 6 min, respectively. L-Selenomethionine appeared rapidly in plasma after administration of D-selenomethionine, whereas D-selenomethionine was not detected in plasma after administration of L-selenomethionine. The fraction of conversion of D-selenomethionine to L-selenomethionine was estimated to be 61.3 ± 14.5%. The present method evaluates the stereoselective pharmacokinetics of selenomethionine enantiomers, including the estimation of the metabolic chiral inversion.