Recent progress in the development of small-molecule fluorescent probes for detection and imaging of selenocysteine and application in thyroid disease diagnosis.

Selenocysteine (SeCys) is the 21st genetically encoded amino acid present in proteins and is involved in various biological functions. Inappropriate levels of SeCys can be considered as a sign of various diseases. Therefore, small molecular fluorescent probes for the detection and imaging of SeCys in vivo in biological systems are considered to be of significant interest for understanding the physiological role of SeCys. Thus, this article mainly provides a critical evaluation of recent advances made in SeCys detection along with the biomedical applications based on small molecular fluorescent probes published in the literature during the past half a dozen years. Therefore, the article primarily deals with the rational design of fluorescent probes, wherein these were selective towards SeCys over other biologically abundant molecules, in particular the thiol-based ones. The detection has been monitored by different spectral techniques, such as fluorescence and absorption spectroscopy and in some cases even visual color changes. Further, the detection mechanism and the utility of fluorescent probes for in vitro and in vivo cell imaging applications are addressed. For clarity, the main features have been conveniently divided into four categories based on the chemical reactions of the probe, viz., in terms of the cleavage of the responsive group by the SeCys nucleophile: (i) 2,4-dinitrobene sulphonamide group, (ii) 2,4-dinitrobenesulfonate ester group, (iii) 2,4-dinitrobenzeneoxy group and (iv) miscellaneous types. Overall this article deals with the analysis of more than two dozen fluorescent probes demonstrated for selective detection of SeCys along with their applications towards disease diagnosis.

Effect of the selenized yeast added in feed on selenium-containing proteins of albumins in egg yolk.

This work was aimed to study the effects of the selenized yeast added in feed on selenium-containing proteins of egg yolk. Two groups of the same little hens were given the ordinary grain feed either unsupplemented selenized yeast (Group O) or supplemented with 0.15% selenized yeast (Group Y), respectively. The water-soluble Se-containing proteins were isolated and purified from the two group eggs yolk using the same conditions. SeP1-1 and SeP1-I were purified from the yolk of Group Y and Group O, respectively. Sequences identified by HPLC-MS/MS showed that SeP1-1 was a highly homologous Se-containing protein with Se-free YGP-42 with 83% match, in which Se species include methylselenocysteine and selenocysteine. SeP1-I was a highly homologous Se-containing protein with Se-free ovalbumin with 78.2% match, in which Se species include selenomethionine and selenocysteine. It can be concluded that the selenized yeast can change the compositions and structures of Se-containing proteins in egg yolk.

Integrated Top-Down and Bottom-Up Mass Spectrometry for Characterization of Diselenide Bridging Patterns of Synthetic Selenoproteins.

With the rapid acceleration in the design and development of new biotherapeutics, ensuring consistent quality and understanding degradation pathways remain paramount, requiring an array of analytical methods including mass spectrometry. The incorporation of non-canonical amino acids, such as for synthetic selenoproteins, creates additional challenges. A comprehensive strategy to characterize selenoproteins should serve dual purposes of providing sequence confirmation and mapping of selenocysteine bridge locations and the identification of unanticipated side products. In the present study, a combined approach exploiting the benefits of both top-down and bottom-up mass spectrometry was developed. Both electron-transfer/higher-energy collision dissociation and 213 nm ultraviolet photodissociation were utilized to provide complementary information, allowing high quality characterization, localization of diselenide bridges for complex proteins, and the identification of previously unreported selenoprotein dimers.

Peanut selenium distribution, concentration, speciation, and effects on proteins after exogenous selenium biofortification.

Fortification of Se is vital importance for both nutritional demand and prevention of Se-deficiency-related diseases. To better understand t selenium distribution, concentration, speciation, its effects on proteins, and cytotoxic activity, the biofortification of exogenous Se in peanut was conducted in this study. Our data have shown that foliar spraying of Se-riched fertilizer was more efficient for biotransformation of inorganic Se to organic Se by peanut plant. Besides, the Se content in peanut was increased in a dose-dependent manner. Our present study also confirmed that SeCys2, MeSeCys, and SeMet were the main Se speciation within peanut proteins. Moreover, the secondary structure and thermostability of peanut protein were altered as a result of the Se treatments, and these alterations could be attributed to the replacements of cysteine and methionine by selenocysteine and selenomethionine, respectively. The Se-enriched peanut protein could significantly inhibit the growth of Caco-2 and HepG2 in a concentration-dependent manner.

Green and simple extraction of free seleno-amino acids from powdered and lyophilized milk samples with natural deep eutectic solvents.

Natural deep eutectic solvents (NADES) were introduced for the extraction of free seleno-amino acids from lyophilized and powdered milk samples. Different NADES were evaluated, and lactic acid:glucose (LGH) showed the highest selenium recoveries. Selenium analysis was performed by inductively coupled plasma mass spectrometry (ICP MS). Se-NADES analysis in ICP MS was optimized according to the radio frequency power and nebulization gas flow rate. Se-NADES extraction was optimized by an experimental design. LGH dilution, LGH volume, sample quantity, and ultrasound time were factors influencing the extraction. Seleno-amino acids were determined by liquid chromatography-ICP MS. After optimization, the limits of detection obtained were 7.37, 8.63, and 9.64 µg kg-1 for selenocysteine, selenomethionine, and seleno-methyl-selenocysteine, respectively. The NADES-extraction is a green procedure with 2 penalty points in the EcoScale. The method was applied to the analysis of powdered milk, lyophilized Se-fortified sheep milk, and ERM-BD151 skimmed milk powder.

A review of bioselenol-specific fluorescent probes: Synthesis, properties, and imaging applications.

Bioselenols are important substances for the maintenance of physiological balance and offer anticancer properties; however, their causal mechanisms and effectiveness have not been assessed. One way to explore their physiological functions is the in vivo detection of bioselenols at the molecular level, and one of the most efficient ways to do so is to use fluorescent probes. Various types of bioselenol-specific fluorescent probes have been synthesized and optimized using chemical simulations and by improving biothiol fluorescent probes. Here, we review recent advances in bioselenol-specific fluorescent probes for selenocysteine (Sec), thioredoxin reductase (TrxR), and hydrogen selenide (H2Se). In particular, the molecular design principles of different types of bioselenols, their corresponding sensing mechanisms, and imaging applications are summarized.

A novel HPLC column switching method coupled to ICP-MS/QTOF for the first determination of selenoprotein P (SELENOP) in human breast milk.

In this work, we describe for the first time the presence of selenoprotein P in human breast milk. To this end, a novel analytical method has been developed based on a two-dimensional column switching system, which consisted of three size exclusion columns and one affinity column coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method combines the accurate quantification of selenoproteins and selenometabolites by species unspecific isotopic dilution ICP-MS, with unequivocal identification by quadrupole-time-of-flight mass spectrometry. Several selenopeptides, which contain the amino acid selenocysteine (U, SeCys), were identified after tryptic digestion followed by their separation. The results reveal that the relative selenium concentration in colostrum follows the order: glutathione peroxidase (GPX) ≈ selenoprotein P (SELENOP) > selenocystamine (SeCA) > other selenometabolites (SeMB), in contrast with previously published papers (GPX > SeCA > selenocystine > selenomethionine). A mean concentration of 20.1 ± 1.0 ng Se g-1 as SELENOP (1.45 µg SELENOP/g) was determined in colostrum (31% of total selenium).

Au nanoparticle-based probe for selenol in living cells and selenium-rich tea and rice.

Selenocysteine (Sec) is a primary kind of reactive selenium species in cells, and its vital roles in physiological processes have been characterized. Therefore, the highly effective method for sensing Sec in metabolic processes and selenium-rich food must be developed. This study presents a new fluorescent probe, namely, GSH-NB@AuNPs, for highly selective detection of selenol based on the fluorescence quenching quality on the surface of gold nanoparticles (AuNPs). The probe consists of glutathione (GSH) and Nile blue (NB) moieties assembled on AuNPs. The probe exhibits excellent sensitivity and selectivity for Sec and is applied in imaging endogenous and exogenous Sec in living cells through confocal fluorescence microscopy. The proposed probe provides a promising and powerful method for detecting selenol in foodstuff (such as selenium-rich rice and tea) with the detection limit of 9.5 nM.

Identification and determination of selenocysteine, selenosugar, and other selenometabolites in turkey liver.

Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. To further study selenometabolites in Se-deficient, Se-adequate, and high-Se liver, turkey poults were fed 0, 0.4, and 5 µg Se g-1 diet as Na2SeO3 (Se(iv)) in a Se-deficient (0.005 µg Se g-1) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. No selenomethionine (SeMet) was detected in liver in turkeys fed either this true Se-deficient diet or supplemented with inorganic Se, showing that turkeys cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient liver, as expected in animals with negligible selenoprotein levels. Sec content in high Se liver only doubled as compared to Se-adequate liver, indicating that the 6-fold increase in liver Se was not due to increases in selenoproteins. What increased dramatically in high Se liver were low molecular weight (MW) selenometabolites: glutathione-, cysteine- and methyl-conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). Substantial Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide bonds. In high-Se liver, these "selenosugar-decorated" proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to low MW selenometabolites. In summary, more Se is present as the selenosugar moiety in Se-adequate liver, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols.

A TP-FRET-based fluorescent sensor for ratiometric visualization of selenocysteine derivatives in living cells, tissues and zebrafish.

Selenium is a biologically essential micronutrient element serving as an essential building block for selenoproteins (SePs), which is playing a key role in various cellular functions. Hence, it is of great significance to developing a reliable and rapid method for detection of Sec in biosystems. Compared with the previously reported probes that have been developed for selective detection of Sec, two-photon (TP) ratiometric Sec-specific probes would be advantageous for the NIR excitation and built-in correction of the dual emission bands. To quantitatively and selectively detect Sec over biothiols with rapid and sensitive response, we for the first time report a new fluorescence resonance energy transfer (FRET)-based TP ratiometric fluorescence probe CmNp-Sec, which was constructed by conjugating a TP fluorophore 6 (coumarin derivative with a D-π-A-structure) with a naphthalimide fluorophore 9 via a non-conjugated linker, and employed a 4-dinitrobenzene-ether (DNB) with a strong ICT effect as Sec responsive moiety. It exhibits quantitatively detect Sec in a wide range (0-50 µM) with a limit of detection of 7.88 nM within 10 min. More impressively, this probe can be conveniently used to detect Sec in living cells, tissues and zebrafish, demonstrating it has the latent capability in further biological applications.

Detection of Selenocysteine with a Ratiometric near-Infrared Fluorescent Probe in Cells and in Mice Thyroid Diseases Model.

The pathological progression of thyroid diseases poses a serious threat to human health. Because thyroid diseases are closely related to selenocysteine (Sec), it is necessary to investigate the relationship between Sec and thyroid diseases. Herein, we design and synthesize a ratiometric near-infrared fluorescent probe (Mito-Cy-Sec) to analyze the fluctuations and roles of Sec in cells and in mice thyroid diseases model. The probe is composed of a near-infrared heptamethine cyanine fluorophore, an acrylamide as the response moiety, and a lipophilic triphenylphosphonium cation as the mitochondrial localization group. After reacting with Sec for 5 min, the probe Mito-Cy-Sec exhibits a distinct ratiometric fluorescence signal accompanied by a color change from green to blue. The applicability of Mito-Cy-Sec in mitochondrial localization is assessed via the super-resolution imaging. Mito-Cy-Sec has been successfully applied to detect the fluctuations of Sec concentration in human thyroid epithelial/cancer cell lines (Nthy-ori-3 cells/BHT101 cells) and mice thyroid disease (thyroiditis and thyroid cancer) models. Besides, both of our probes Mito-Cy-Sec and commercial ROSGreen H2O2 are employed to examine the interrelationship between H2O2 and Sec in cells and in mice models. The results demonstrate that the relevant-levels between H2O2 and Sec are exactly negative correlation. The related-levels of Sec and H2O2 may be identified as diagnostic indicators for the auxiliary diagnosis of thyroid diseases. We suppose that our probe Mito-Cy-Sec can be employed as a promising chemical tool for the diagnosis of thyroid diseases.

Bioluminescence Imaging of Selenocysteine in Vivo with a Highly Sensitive Probe.

Selenocysteine (Sec), a vital member of reactive selenium species, is closely implicated in diverse pathophysiological states, including cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and male infertility. Monitoring Sec in vivo is of significant interest for understanding the physiological roles of Sec and the mechanisms of human diseases associated with abnormal levels of Sec. However, no bioluminescence probe for real-time monitoring of Sec in vivo has been reported. Herein, we present a novel bioluminescent probe BF-1 as an effective tool for the determination of Sec in living cells and in vivo for the first time. BF-1 has advantages of high sensitivity (a detection limit of 8 nM), remarkable bioluminescence enhancement (580-fold), reasonable selectivity, low cytotoxicity, and high signal-to-noise ratio imaging feasibility of Sec in living cells and mice. More importantly, BF-1 affords high sensitivity for monitoring Sec stimulated by Na2SeO3 in tumor-bearing mice. These results demonstrate that our new probe could serve as a powerful tool to selectively monitor Sec in vivo, thus providing a valuable approach for exploring the physiological and pathological functions and anticancer mechanisms of selenium.

Ultra-High Resolution Elemental/Isotopic Mass Spectrometry (m/Δm > 1,000,000): Coupling of the Liquid Sampling-Atmospheric Pressure Glow Discharge with an Orbitrap Mass Spectrometer for Applications in Biological Chemistry and Environmental Analysis.

Many fundamental questions of astrophysics, biochemistry, and geology rely on the ability to accurately and precisely measure the mass and abundance of isotopes. Taken a step further, the capacity to perform such measurements on intact molecules provides insights into processes in diverse biological systems. Described here is the coupling of a combined atomic and molecular (CAM) ionization source, the liquid sampling-atmospheric pressure glow discharge (LS-APGD) microplasma, with a commercially available ThermoScientific Fusion Lumos mass spectrometer. Demonstrated for the first time is the ionization and isotopically resolved fingerprinting of a long-postulated, but never mass-spectrometrically observed, bi-metallic complex Hg:Se-cysteine. Such a complex has been implicated as having a role in observations of Hg detoxification by selenoproteins/amino acids. Demonstrated as well is the ability to mass spectrometrically-resolve the geochronologically important isobaric 87Sr and 87Rb species (Δm ~ 0.3 mDa, mass resolution m/Δm ≈ 1,700,000). The mass difference in this case reflects the beta-decay of the 87Rb to the stable Sr isotope. These two demonstrations highlight what may be a significant change in bioinorganic and atomic mass spectrometry, with impact expected across a broad spectrum of the physical, biological, and geological sciences. Graphical Abstract "".

Nutraceutical formulation, characterisation, and in-vitro evaluation of methylselenocysteine and selenocystine using food derived chitosan:zein nanoparticles.

Selenoamino acids (SeAAs) have been shown to possess antioxidant and anticancer properties. However, their bioaccessibility is low and they may be toxic above the recommended nutritional intake level, thus improved targeted oral delivery methods are desirable. In this work, the SeAAs, Methylselenocysteine (MSC) and selenocystine (SeCys2) were encapsulated into nanoparticles (NPs) using the mucoadhesive polymer chitosan (Cs), via ionotropic gelation with tripolyphosphate (TPP) and the NPs produced were then coated with zein (a maize derived prolamine rich protein). NPs with optimized physicochemical properties for oral delivery were obtained at a 6: 1 ratio of Cs:TPP, with a 1:0.75 mass ratio of Cs:zein coating (diameter ~260 nm, polydispersivity index ~0.2, zeta potential >30 mV). Scanning Electron Microscopy (SEM) analysis showed that spheroidal, well distributed particles were obtained. Encapsulation Efficiencies of 80.7% and 78.9% were achieved, respectively, for MSC and SeCys2 loaded NPs. Cytotoxicity studies of MSC loaded NPs showed no decrease in cellular viability in either Caco-2 (intestine) or HepG2 (liver) cells after 4 and 72 h exposures. For SeCys2 loaded NPs, although no cytotoxicity was observed in Caco-2 cells after 4 h, a significant reduction in cytotoxicity was observed, compared to pure SeCys2, across all test concentrations in HepG2 after 72 h exposure. Accelerated thermal stability testing of both loaded NPs indicated good stability under normal storage conditions. Lastly, after 6 h exposure to simulated gastrointestinal tract environments, the sustained release profile of the formulation showed that 62 ±â€¯8% and 69 ±â€¯4% of MSC and SeCys2, had been released from the NPs respectively.

Investigation of free and conjugated seleno-amino acids in wheat bran by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

An analytical method for determining seleno-methionine, methyl-seleno-cysteine, and seleno-cystine in wheat bran was developed and validated. Four different extraction procedures were evaluated to simultaneously extract endogenous free and conjugated seleno-amino acids in wheat bran in order to select the best extraction protocol in terms of seleno amino acid quantitation. The extracted samples were subjected to a clean-up by a reversed phase/strong cation exchange solid-phase extraction and analyzed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. The optimized extraction protocol was employed to validate the methodology. Process efficiency ranged from 58 to 112% and trueness from 73 to 98%. Limit of detection and limit of quantification were lower than 1 ng/g. Four wheat bran samples were analyzed for both total Se and single seleno-amino acids determination. The results showed that Se- seleno-methyl-lselenocysteine was the major seleno-amino acid in wheat bran while seleno-methionine and seleno-cysteine were both minor species.

Assessing bioaccessibility of Se and I in dual biofortified radish seedlings using simulated in vitro digestion.

Selenium (Se) and iodine (I) are essential elements for humans, and biofortification of vegetables with these elements is an effective way to amend their deficiencies in the diet. In this study, the distribution and transformation of Se and I species were investigated in radish seedlings that were simultaneously supplemented with these two elements; the fate and the bioaccessibility of Se and I species were dynamically surveyed in the oral, gastric and intestinal phases using a simulated in vitro digestion method. The radish seedlings were cultivated in hydroponic conditions with Se (IV), Se (VI), I- and IO3- (each 1 mg L-1). The results revealed that Se-methylselenocysteine (MeSeCys), selenocystine (SeCys2), selenomethionine (SeMet) and Se (VI) were present in radish, and MeSeCys was the dominant species in both gastric and intestinal extracts, comprising 32.7 ±â€¯1.5% and 39.6 ±â€¯1.1% of the total content, respectively. I- was also the dominant species, which accounted for 57.1 ±â€¯2.1%, 46.6 ±â€¯1.5% and 68.8 ±â€¯1.8% of the total digested content respectively in the oral, gastric and intestinal extracts. Meanwhile, IO3- was absent and organic I accounted for approximately 20%. The bioaccessibility of Se and I in the intestinal phase reached 95.5 ±â€¯2.5% and 85.8 ±â€¯0.9%, respectively; although after dialysis through membranes, the data reduced to 60.1 ±â€¯2.8% and 39.6 ±â€¯0.8%, respectively. Contents of MeSeCys and I- increased from the oral to intestinal phase and the bioaccessibility of both Se and I in radish was above 85%. So radish is suitable as a potential dietary source of Se and I with biofortification.

In vitro bioaccessibility of selenoamino acids from selenium (Se)-enriched Chlorella vulgaris biomass in comparison to selenized yeast; a Se-enriched food supplement; and Se-rich foods.

Selenium (Se) is an indispensable microelement in our diet and health issues resulting from deficiencies are well documented. Se-containing food supplements are available on the market including Se-enriched Chlorella vulgaris (Se-Chlorella) which accumulates Se in the form of Se-amino acids (Se-AAs). Despite its popular uses, data about the bioaccessibility of Se-AAs from Se-Chlorella are completely missing. In the present study, gastrointestinal digestion times were optimized and the in vitro bioaccessibility of Se-AAs in Se-Chlorella, Se-yeast, a commercially available Se-enriched food supplement (Se-supplement) and Se rich foods (Se-foods) were compared. Higher bioaccessibility was found in Se-Chlorella (∼49%) as compared to Se-yeast (∼21%), Se-supplement (∼32%) and Se-foods. The methods used in production of Se-Chlorella biomass were also investigated. We found that disintegration increased bioaccessibility whereas the drying process had no effect. Similarly, temperature treatment by microwave oven also increased bioaccessibility whereas boiling water did not.

Investigation of free seleno-amino acids in extra-virgin olive oil by mixed mode solid phase extraction cleanup and enantioselective hydrophilic interaction liquid chromatography-tandem mass spectrometry.

An analytical method for determining seleno-methionine (SeMet), methyl-seleno-cysteine and seleno-cystine in extra-virgin olive oil (EVOO) was developed and validated. EVOO sample (15 g) was diluted with hexane, extracted with methanol/water 80:20 (v/v), and cleaned up by a reversed phase/strong cation exchange solid phase extraction. Analysis was performed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. Process efficiency ranged between 49 and 97% and trueness between 87 and 126%, with intermediate precision, expressed as standard deviation, lower than 10%. Method detection limits (MDLs) and method quantification limits (MQLs) were lower than 1 µg kg-1. Thirty-two EVOO samples from different Italian regions were analyzed for both total Se and single seleno-amino acids determination. Only l-SeMet was found at level MQL (0.2 µg kg-1)-1.42 µg kg-1 in ten samples, while total Se was in the range of MDL-9.1 µg kg-1. Concentration of l-SeMet (5-6% of total Se) and total Se correlated very well to each other (R2 = 0.995).

Selenocysteine-Specific Mass Spectrometry Reveals Tissue-Distinct Selenoproteomes and Candidate Selenoproteins.

Selenoproteins, defined by the presence of selenocysteines (Sec), play important roles in a wide range of biological processes. All known selenoproteins are marked by the presence of Sec insertion sequence (SECIS) at their mRNA. The lack of an effective analytical method has hindered our ability to explore the selenoproteome and new selenoproteins beyond SECIS. Here, we develop a Sec-specific mass spectrometry-based technique, termed "SecMS," which allows the systematic profiling of selenoproteomes by selective alkylation of Sec. Using SecMS, we quantitatively characterized the age- and stress-regulated selenoproteomes for nine tissues from mice of different ages and mammalian cells, demonstrating tissue-specific selenoproteomes and an age-dependent decline in specific selenoproteins in brains and hearts. We established an integrated platform using SecMS and SECIS-independent selenoprotein (SIS) database and further identified five candidate selenoproteins. The application of this integrated platform provides an effective strategy to explore the selenoproteome independent of SECIS.

In vitro bioaccessibility and speciation changes of selenium in Pleurotus eryngii during the growing stage.

The production of Pleurotus eryngii by selenium (Se) biofortification is an effective way to improve the demand for Se in humans. In order to investigate the Se bioaccessibility and speciation of Se-enriched P. eryngii during the growing stage, the Se distribution in biochemical fractions, and the molecular weight and the Se species of Se-containing compounds derived from in vitro simulated gastrointestinal fluids were analyzed by size exclusion and anion exchange-high performance liquid chromatography and inductively coupled plasma mass spectrometry. The results showed that albumin had the highest Se content among biochemical fractions, approximately 34.40% of total Se, followed by glutelin, globulin and gliadins. Selenomethionine that was proved to be the major Se species would increase with P. eryngii growing from 45.85% to 59.32%, while selenocysteine would decrease from 40.68% to 15.17% of total Se. In conclusion, selenocysteine would gradually convert to selenomethionine, and thus the bioaccessibility of Se was greater in mature P. eryngii than in younger mushrooms.