An accelerated and optimized algorithm of selenium-encoded isotopic signature targeted profiling for global selenoproteome analysis.

Selenoproteins play crucial roles including protection and recovery from oxidative stress in organisms. Direct profiling of selenoproteins in proteomes is challenging due to their extremely low abundance. We have developed a computational algorithm termed selenium-encoded isotopic signature targeted profiling (SESTAR) to increase the sensitivity of detecting selenoproteins in complex proteomic samples. In this chapter, we briefly described the basic algorithm of SESTAR. We then introduced SESTAR++, an updated version of SESTAR, with accelerated computation speed and lowered false positive rate. We also provided a detailed workflow to apply SESTAR++ to proteomic profiling of selenoproteins, including the instruction of running the software and implementing it in a targeted profiling mode.

Reinjection flow field-flow fractionation method for nanoparticle quantitative analysis in unknown and complex samples.

An analytical challenge that arises in environmental and food analysis is to quantify heterogeneous nanoparticles especially in polydisperse and complex samples. The method stated herein based on the reinjection asymmetrical flow field-flow fractionation (AF4 × AF4) coupled with inductively coupled plasma-mass spectrometer (ICP-MS) and statistical deconvolution allowed for identifying the molecular weight (Mw) and selenium abundance of the low Mw protein fractions (ca. < 132 kDa) in an unknown and complex sample (e.g., selenium-rich soybean protein isolates (Se-SPI)). A non-linear decay crossflow program was also developed to get better resolution and shorter elution time for both low and high Mw components. The concept of the reinjection method was based on the excellent ability for reducing sample complexity regarding the size fractionation, and peak reproducibility under the identical conditions of AF4 system. The standard protein mixture was used as a proof-of-principle sample. The results showed the underlying peaks predicted by the reinjection method were agreed with the separation result using the standard mixture (the relative standard deviation of peak locations < 1%), which indicated the reinjection method could provide an accurate assessment of the underlying peak number and location, and was promising to minimize the overfitting problem for statistic deconvolution. Interestingly, significant differences of Se abundance in protein fractions were observed in the low Mw range for Se-SPI, ranging from 0.28 to 1.66 cps/V with the Mw ranging from 13.75 kDa to 104.17 kDa, which indicated significant differences in the ability of binding Se for these selenium-rich proteins in Se-SPI.

Isolation and identification of three water-soluble selenoproteins in Se-enriched Agaricus blazei Murrill.

Selenoproteins in selenium (Se)-enriched vegetables play an important role in human health. In this study, three water-soluble selenoproteins PR-Se-1, PR-Se-2 and PR-Se-3 in Agaricus blazei Murrill (ABM) were isolated by anion exchange chromatography, gel filtration chromatography and SDS-PAGE. Sequence analyses performed by HPLC-MS/MS showed that PR-Se-1, a 114024 Da selenoprotein with 1019 amino acids (AAs), is an isoenzyme of isocitrate dehydrogenase. PR-Se-2, a 53983 Da selenoprotein with 508 AAs, is a kind of dihydrolipoyl dehydrogenase. PR-Se-3, a 47179 Da selenoprotein with 415 AAs, is a kind d-proline reductase. Se content is high at 26.1 µg/g, and selenocystine is the predominant Se unit in the three selenoproteins. Se content of ABM is 9.15 µg/g, and the organic form of Se accounts for ~81% of total Se content. ABM could be a promising source of Se in Se-poor regions.

The Relevance of Selenium Status in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune and inflammatory disease that can cause joint damage. Among the environmental risk factors, diet plays an important role because it can aggravate or attenuate inflammation. Selenium (Se) is considered an essential trace element since it is a structural component of antioxidant enzymes; however, its concentration can be affected by diet, drugs and genetic polymorphisms. Studies have reported that RA patients have a deficient diet in some food groups that is associated with parameters of disease activity. Furthermore, it has been shown that there is an alteration in serum Se levels in this population. Although some clinical trials have been conducted in the past to analyze the effect of Se supplementation in RA, no significant results were obtained. Contrastingly, experimental studies that have evaluated the effect of novel Se nanoparticles in RA-induced models have shown promising results on the restoration of antioxidant enzyme levels. In particular, glutathione peroxidase (GPx) is an important selenoprotein that could have a modulating effect on inflammation in RA. Considering that RA patients present an inflammatory and oxidative state, the aim of this review is to give an overview of the current knowledge about the relevance of Se status in RA.

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).

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.

Medición del contenido de selenio en especímenes biológicos: aplicación en el laboratorio clínico / Measurement of selenium content in biological specimens: medicine laboratory application

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Foliar application is an effective method for incorporating selenium into peanut leaf proteins with antioxidant activities.

Proteins were extracted from Se-enriched peanut leaves, an agro-byproduct, and the foliar application of sodium selenite was indicated to be an effective method to incorporate Se into leaf selenoproteins with 75-80% incorporation rates. After trypsin digestion, the most abundant proteins from Se-enriched peanut leaf (PSPL) were identified as pathogenesis-related class 10 proteins, Ara h 8 allergen and its isoforms, using LC-MS/MS. The Se species in both the low Se PSPL and high Se PSPL were determined to be selenomethionine (SeMet), methylselenocysteine (MeSeCys) and selenocystine (SeCys2) with SeMet (15.6 mg/g) dominated the high Se PSPL. Their antioxidant activities were also evaluated using free radical scavenging assay, reducing power assay and ferric thiocyanate (FTC) test. As results, the PSPL exhibited potent DPPH radical (96.2%) and superoxide anion radical (98.4%) scavenging activities and showed strong reducing power in a Se-concentration-dependent manner, indicating that PSPL can be used as antioxidants and Se sources to improve health.

Expanding beyond ICP-MS to better understand selenium biochemistry.

Selenium is an essential trace element in human health and therefore its concentration in biological samples (biofluids and tissues) is used as an indicator of health and nutritional status. In humans, selenium's biological activity occurs through the 25 identified selenoproteins. As total selenium concentration encompasses both functional selenoproteins, small selenocompounds and other selenium-binding proteins, selenium speciation, rather than total concentration, is critical in order to assess functional selenium. Previously, quantitative analysis of selenoproteins required laborious techniques that were often slow and costly. However, more recent advancements in tandem mass spectrometry have facilitated the qualitative and quantitative identification of these proteins. In light of the current alternatives for understanding selenium biochemistry, we aim to provide a review of the modern applications of electrospray ionisation mass spectrometry (ESI-MS) as an alternative to inductively coupled plasma mass spectrometry (ICP-MS) for qualitative and quantitative selenium speciation.

Selenoproteome Identification in Inflamed Murine Primary Bone Marrow-Derived Macrophages by Nano-LC Orbitrap Fusion Tribrid Mass Spectrometry.

Selenium (Se) functions as a cellular redox gatekeeper through its incorporation into proteins as the 21st amino acid, selenocysteine (Sec). Supplementation of macrophages with exogenous Se (as sodium selenite) downregulates inflammation and intracellular oxidative stress by effectively restoring redox homeostasis upon challenge with bacterial endotoxin lipopolysaccharide (LPS). Here, we examined the use of a standard Tandem Mass Tag (TMT)-labeling mass spectrometry-based proteomic workflow to quantitate and examine temporal regulation of selenoproteins in such inflamed cells. Se-deficient murine primary bone marrow-derived macrophages (BMDMs) exposed to LPS in the presence or absence of selenite treatment for various time periods (0-20 h) were used to analyze the selenoproteome expression using isobaric labeling and shotgun proteomic workflow. To overcome the challenge of identification of Sec peptides, we used the identification of non-Sec containing peptides downstream of Sec as a reliable evidence of ribosome readthrough indicating efficient decoding of Sec codon. Results indicated a temporal regulation of the selenoproteome with a general increase in their expression in inflamed cells in a Se-dependent manner. Selenow, Gpx1, Msrb1, and Selenom were highly upregulated upon stimulation with LPS when compared to other selenoproteins. Interestingly, Selenow appeared to be one amongst the highly regulated selenoproteins in macrophages that was previously thought to be mainly restricted to myocytes. Collectively, TMT-labeling method of non-Sec peptides offers a reliable method to quantitate and study temporal regulation of selenoproteins; however, further optimization to include Sec-peptides could make this strategy more robust and sensitive compared to other semi-quantitative or qualitative methods. Graphical Abstract.

Proposal of a study protocol of a preliminary double-blind randomized controlled trial. Verifying effects of selenium supplementation on selenoprotein p and s genes expression in protein and mRNA levels in subjects with coronary artery disease: selenegene.

BACKGROUND: Selenium is the component of selenocystein amino acid, which itself is the building block of selenoproteins having diverse effects on various aspects of the human health. Among these proteins, selenoprotein P is the central to the distribution and homeostasis of selenium, and selenoprotein S as a transmembrane protein is associated with a range of inflammatory markers, particularly in the context of cardiovascular disease. It is known that selenium status outside of the normal range is considered to confer different benefits or adverse cardiovascular risk factors. Therefore, for the first time, we aimed to verify effects of Selenium supplementation on Selenoprotein P and S Genes Expression in Protein and mRNA Levels in Subjects with Coronary Artery Disease (CAD). METHODS: This is the study protocol of a double blinded randomized clinical trial on 130 subjects with angiographically documented stenosis of more than 75% in one or more coronary artery vessels. In this 60-day study, 65 patients in each group received either a 200mg selenium yeast or placebo tablets once daily. During the study, subjects were followed by phone calls and visited our clinic twice to repeat baseline measurements. We hypothesized that our finding would enable a more basic and confirmed understanding for the effect of selenium supplementation by investigating its effect on gene expression levels in people with CAD. DISCUSSION: Upon confirmation of this hypothesis, the beneficial effect of inflammation regulation by supplementation with micronutrients could be considered for subjects with CVD.

Participation of selenoproteins localized in the ER in the processes occurring in this organelle and in the regulation of carcinogenesis-associated processes.

The functions performed by the ER are diverse: synthesis of steroid hormones, synthesis of proteins for the plasma membrane, lysosomes, as well as proteins meant for exocytosis, protein folding, formation of disulfide bonds, N-linked glycosylation, etc. Selenoproteins localized in this organelle are definitely involved in the processes occurring in it, and the most common of them include participation in protein degradation, regulation of ER stress and redox metabolism. ER stress has been registered in many types of cancer cells. The ability to persist under prolonged ER stress increases their survival, resistance to drugs and immunity. Disturbances in the redox regulation of the cell cycle, which result in the accumulation of misfolded proteins in the ER, viral infection, disruption of Ca2+ regulation, are known to cause an evolutionarily conserved reaction - unfolded protein response (UPR) and, ultimately, lead to ER stress. Since selenoproteins, as oxidoreductases, possess antioxidant properties, and their role in the regulation of important processes, such as carcinogenesis and ER stress, has been actively studied in the recent decades, the subject of this review is highly relevant.

Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis?

BACKGROUND: Exosomes isolated from plasma of patients with sepsis may induce vascular apoptosis and myocardial dysfunction by mechanisms related to inflammation and oxidative stress. Despite previous studies demonstrating that these vesicles contain genetic material related to cellular communication, their molecular cargo during sepsis is relatively unknown. In this study, we evaluated the presence of microRNAs (miRNAs) and messenger RNAs (mRNAs) related to inflammatory response and redox metabolism in exosomes of patients with septic shock. METHODS: Blood samples were collected from 24 patients with septic shock at ICU admission and after 7 days of treatment. Twelve healthy volunteers were used as control subjects. Exosomes were isolated by ultracentrifugation, and their miRNA and mRNA content was evaluated by qRT-PCR array. RESULTS: As compared with healthy volunteers, exosomes from patients with sepsis had significant changes in 65 exosomal miRNAs. Twenty-eight miRNAs were differentially expressed, both at enrollment and after 7 days, with similar kinetics (18 miRNAs upregulated and 10 downregulated). At enrollment, 35 differentially expressed miRNAs clustered patients with sepsis according to survival. The pathways enriched by the miRNAs of patients with sepsis compared with control subjects were related mostly to inflammatory response. The comparison of miRNAs from patients with sepsis according to hospital survival demonstrated pathways related mostly to cell cycle regulation. At enrollment, sepsis was associated with significant increases in the expression of mRNAs related to redox metabolism (myeloperoxidase, 64-fold; PRDX3, 2.6-fold; SOD2, 2.2-fold) and redox-responsive genes (FOXM1, 21-fold; SELS, 16-fold; GLRX2, 3.4-fold). The expression of myeloperoxidase mRNA remained elevated after 7 days (65-fold). CONCLUSIONS: Exosomes from patients with septic shock convey miRNAs and mRNAs related to pathogenic pathways, including inflammatory response, oxidative stress, and cell cycle regulation. Exosomes may represent a novel mechanism for intercellular communication during sepsis.

Nonradioactive Isotopic Labeling and Tracing of Selenoproteins in Cultured Cell Lines.

Selenium (Se) is an essential component of genetically encoded selenoproteins, in the form of a rare amino acid, namely the selenocysteine (Sec). Radioactive 75Se has been widely used to trace selenoproteins in vitro and in vivo (cell models and animals). Alternatively, its unique isotopic pattern can be used to detect and characterize nonradioactive Se-compounds in cellular extracts using molecular or elemental mass spectrometry at ppm levels. However, when studying trace levels of Se-compounds, such as selenoproteins (ppt levels), the distribution of the signal between its six naturally abundant isotopes reduces its sensitivity. Here, we describe the use of isotopically enriched forms of Se as an alternative strategy to radioactive 75Se, for the labeling and tracing of selenoproteins in cultured cell lines.

Radioactive 75Se Labeling and Detection of Selenoproteins.

The trace element selenium (Se) is incorporated into proteins as the amino acid selenocysteine (Sec), which is cotranslationally inserted into specific proteins in response to a UGA codon. Proteins containing Sec at these specific positions are called selenoproteins. Most selenoproteins function as oxidoreductases, while some serve other important functions. There are 25 known selenoprotein genes in humans and 24 in mice. The use of Sec allows selenoproteins to be detected by a convenient method involving metabolic labeling with 75Se. Labeling of cells and whole animals are used for the examination of selenoprotein expression profiles and the investigation of selenoprotein functions. In mammals, nonspecific 75Se insertion is very low, and sensitivity and specificity of selenoprotein detection approaches that of Western blotting. This method allows for the examination of selenoprotein expression and Se metabolism in model and non-model organisms. Herein, we describe experimental protocols for analyzing selenoproteins by metabolic labeling with 75Se both in vitro and in vivo. As an example, the procedure for metabolic labeling of HEK293T human embryonic kidney cells is described in detail. This approach remains a method of choice for the detection of selenoproteins in diverse settings.

Detection of Selenoproteins by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP MS) in Immobilized pH Gradient (IPG) Strips.

In contrast to other trace elements that are cofactors of enzymes and removed from proteins under denaturing conditions, Se is covalently bound to proteins when incorporated into selenoproteins, since it is a component of selenocysteine aminoacid. It implies that selenoproteins can undergo several biochemical separation methods in stringent and chaotropic conditions and still maintain the presence of selenium in the primary sequence. This feature has been used to develop a method for the detection of trace levels of human selenoproteins in cell extracts without the use of radioactive isotopes. The selenoproteins are separated as a function of their isoelectric point (pI) using iso-electrofocusing (IEF) electrophoretic strips and detected by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). This method, therefore referred to as IEF-LA-ICP MS, allowed the detection of several selenoproteins in human cell lines, including Gpx1, Gpx4, TXNRD1, TXNRD2, and SELENOF.

Imaging of Selenium by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) in 2-D Electrophoresis Gels and Biological Tissues.

Selenium and selenoproteins are important components of living organisms that play a role in different biological processes. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a powerful analytical technique that has been employed to obtain distribution maps of selenium in biological tissues in a direct manner, as well as in selenoproteins, previously separated by their molecular masses and isoelectric points using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In this chapter, we present the protocols to perform LA-ICP-MS imaging experiments, allowing the distribution visualization and determination of selenium and/or selenoproteins in biological systems.

Toenail selenium as an indicator of environmental exposure: A cross-sectional study.

The relation between toxicity and essentiality of selenium (Se) is of growing interest in human health, as the effects may widely differ depending of its different chemical species and the exposure levels. Toenail Se has been proposed as a reliable biomarker of long-term Se exposure, but few studies investigated the correlation between its toenail content and environmental determinants (i.e., dietary food intake). We aimed to determine the relation of toenail Se levels with serum Se species as well as food items. We recruited a random sample of Modena (Northern Italy) municipal residents, from whom we collected detailed personal information, dietary habits, toenail specimen for Se determination and a blood sample for serum Se speciation analysis. Toenail Se mean value was 0.96 µg/g (range, 0.47­1.60), with slightly higher levels in females, in non-obese subjects and in Se supplements users, while it was lower in current smokers. Toenail Se positively correlated with organic Se forms, mainly selenoprotein P and selenocysteine, and inversely with the inorganic forms (selenite and selenate). Toenail Se was not associated with meat, cereals and dairy products consumption, positively correlated with fruit and slightly with vegetable intake, and negatively with fish and seafood consumption. Finally, no clear association emerged with estimated air Se exposure.

Selenium Deficiency Mainly Influences Antioxidant Selenoproteins Expression in Broiler Immune Organs.

Selenoprotein has many functions in chicken, and the expression of selenoproteins is closely associated with the selenium (Se) level. However, little is known about the expression patterns of selenoproteins in chicken immune organs. Here, we investigated the effect of dietary Se deficiency on the expressions of 23 selenoproteins in broiler immune organs. In this study, 150 broilers were randomly divided into two groups (75 chickens per group). The chickens were maintained either on a diet supplemented with Se through the addition of 0.2 mg/kg of Se (C group) via sodium selenite or on a Se-deficient granulated diet (L group) until the broilers exhibited an onset of exudative diathesis (ED). Following euthanasia, the samples from the immune tissues (including the spleen, thymus, and bursa of Fabricius) were quickly collected, and the messenger RNA (mRNA) expression levels of 23 selenoproteins were examined by real-time quantitative PCR and analyzed using principal component analysis. The results showed that Se deficiency decreased the mRNA levels of 23 selenoproteins in the thymus, spleen, and bursa of the Fabricius tissues of broiler chickens. Furthermore, we found that among 23 selenoproteins, the mRNA levels of Dio1 in the thymus, Txnrd2 in the spleen, and Txnrd3 in the bursa of Fabricius decreased significantly (90.9 %, 83.3 %, and 96.8 %, respectively). In addition, the principal component analysis (PCA) results suggested that Se deficiency mainly influenced the expression of antioxidative selenoproteins, especially glutathione peroxidases (Gpxs), thioredoxin reductases (Txnrds), and iodothyronine deiodinases (Dios) in chicken immune organs. The results of this study are valuable for understanding the relevance of selenoprotein activity in vivo.

Selenium, selenoproteins and neurodegenerative diseases.

It is unsurprising that our understanding of the role of selenium in neurological function is somewhat immature, considering its relatively recent discovery as an essential element to human health. Selenocysteine, the 21st amino acid, is the defining feature of the 25 selenoprotein-encoding genes so far discovered within the human genome. The low abundance of these proteins in the brain belies the integral role they play in normal neurological function, from well-characterised antioxidant activity in the periphery to poorly understood mechanisms that modulate mitochondrial function and response to brain pathology. Selenium has been identified as playing a role in several neurodegenerative disorders, including Alzheimer's and Parkinson's disease, though its function as a 'cause or effect' of disease process remains unclear. This review discusses selenium metabolism in detail, specifically with regard to the role it plays within the central nervous system, and examines the most current literature investigating how selenium may be involved in chronic diseases of the central nervous system.