Development of new analytical methods for selenium speciation in selenium-enriched yeast material

A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material. The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns. It showed that water-soluble selenium is present in several fractions with a large mass distribution. Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments. The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis. Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography. The study revealed the existence of various unidentified Se species in yeast material. The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS. This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands.     

Identification of selenoproteins in the endoplasmatic reticulum of the rat kidney by combination of radioanalytical techniques and biochemical methods

Investigations have been carried out on rats to obtain information about the selenium-containing proteins present in the microsomal fraction and especially in the endoplasmatic reticulum (ER) of the kidney. For the determination of the selenium levels instrumental neutron activation analysis via ⁷⁵Se was used. After labeling of rats in vivo with ⁷⁵Se-selenite and separation of the proteins in the renal homogenate and cell compartments by electrophoretic methods, the ⁷⁵Se-containing proteins were detected by autoradiography. In this way, six selenium-containing proteins with molecular masses of 15, 16, 20, 23-25, 40-42 and 58-60 were found in the endoplasmatic reticulum. All of those were characterized as selenocysteine-containing selenoproteins. This revised version was published online in August 2006 with corrections to the Cover Date.     

Native protein mapping and visualization of protein interactions in the area of human plasma high‐density lipoprotein by combining nondenaturing micro 2DE and quantitative LC‐MS/MS

A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high‐density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC‐MS/MS. Grid‐cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC‐MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A‐I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A‐I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A‐I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions.     

Identification of selenium-containing proteins in selenium-rich yeast aqueous extract by 2D gel electrophoresis, nanoHPLC-ICP MS and nanoHPLC-ESI MS/MS

An approach based on the consecutive use of nanoHPLC-ICP collision cell MS and nanoHPLC-electrospray MS was proposed for the analysis of water-soluble selenium-containing proteins in selenium-rich yeast after their separation by 2D gel electrophoresis (GE). An ultrasonic probe was employed for fast protein extraction avoiding sample heating and thus reducing the risk of protein degradation. The efficiency of different extraction steps were critically evaluated by total selenium analysis and size-exclusion chromatography (SEC)-ICP MS. Prior to electrophoresis proteins were purified by acetone precipitation. The protein-containing spots from 2D GE were excised and digested with trypsin. The digests obtained were analyzed by nanoHPLC-ICP MS in order to check for the presence of selenium-containing peptides; this allowed the detection of target proteins for further analyses (two out of five spots). The subsequent analyses of the selected digests by nanoHPLC-ES MS/MS allowed the attribution of amino acid sequences to peaks detected by ICP MS revealing the presence of two selenium-containing proteins: SIP 18 and HSP 12.     

Studies on the selenoproteome in the cultured cells of lung and trachea by gel electrophoretic techniques

In the present studies radiotracer techniques have been combined with biochemical separation procedures to investigate the selenium-containing proteins in the culture cells of the lung, trachea and their subcellular fractions. Subcellular separation of the lung and trachea tissues has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of lung and trachea cultured cells in vitro with Se-75, gel electrophoretic separation of the proteins and autoradiographic detection of the tracer. The protein separation by gel electrophoresis using mono-dimensional (1D)- and two-dimensional (2D)-SDS-PAGE has been successfully applied for the selenium research. It has resulted in the detection of a large number of selenium-containing proteins. Two-dimensional gel electrophoresis (2-DE) was also helpful in the identification of the proteins of interest according to their molecular mass and isoelectric point. In this way more than 30 selenium-containing proteins could be distinguished in the lung and trachea samples. Some of them such as Gpx1, Trx1, SelP, SelT and Sel15 could be identified by means of immunoassays, their molecular weight and pI values and localized in the cellular compartments.     

Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC‐MS/MS

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB‐stained spots were numbered and subjected to in‐gel digestion and quantitative LC‐MS/MS. The analysis provided the assignment of 1–25 (average eight) non‐redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC‐MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well‐known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis.     

Selenium bioaccessibility assessment in selenized yeast after "in vitro" gastrointestinal digestion using two-dimensional chromatography and mass spectrometry

In order to investigate the potentially bioavailable selenium-containing compounds in the selenized yeast candidate reference material SEAS 6, a two-dimensional (size exclusion-reversed phase) chromatography approach has been worked out. Electrospray tandem mass spectrometry (ESI Q-TOF MS) was then used for off-line identification of low molecular weigh selenocompounds generated during the gastrointestinal digestion. Selenomethionine (SeMet) was the major compound identified in the gastrointestinal extract while SeMet selenoxide was its main degradation product formed after medium and long-term sample storage, respectively. Total Se and SeMet were quantified in both the soluble extracts and the residue. Results showed that 89+/-3% of total Se was extracted after gastrointestinal digestion, but only 34+/-1% was surprisingly quantified as free SeMet. The rest of Se was present as many other low, medium and high molecular weight Se-species, which could be detected and further characterized by using the two-dimensional chromatography approach proposed here. Interestingly, most of Se-species seemed to be Se-peptides unspecifically produced by the gastrointestinal juice. These results show for the first time that while the efficiency of human gastrointestinal digestion to dissolve Se-containing proteins present in yeast may be high, its efficiency to convert them into free SeMet is much lower. Se-species present in the insoluble residue (not assimilated by the organism), accounting for 11+/-1% of the total Se in selenized yeast, were also studied. After treatment with SDS (denaturing agent) only 13+/-2% of this "insoluble" Se was solubilized, indicating that it was mainly non-protein bound and likely associated to other insoluble matrix components.     

Speciation and subcellular location of Se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic fluorescence spectrometric detection

Speciation of Se-containing proteins in the subcellular fractions of human liver was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by hydride generation-atomic fluorescence spectrometric (HG-AFS) detection. It was found that about 24 kinds of Se-containing proteins existed in subcellular fractions of normal human liver. The molecular weights (MW) of the subunits were mostly in the range 20-30 kDa and 50-80 kDa. Major Se-containing protein fractions at 61 kDa and 21 kDa are probably selenoprotein P and glutathione peroxidase, respectively. The 54 kDa protein is probably a thioredoxin reductase, which is presented in nuclei, mitochondria, lysosome, microsome and cytosol. We noticed that the Se-containing protein with the lowest MW of 9.3 kDa only existed in lysosome. Most of the proteins have not been identified and would require further investigation to characterize them. The specific subcellular distributions of different Se-containing proteins suggest that they could play important biological roles in each organelle.     

Purification and characterization of two major selenium-containing proteins in selenium-rich silkworm pupas

Selenium (Se) is an essential trace element in vivo involved in the defense against oxidative stress. Se deficiency is associated with many human diseases. The bioactivity of Se is dose- and species-dependent. Silkworm pupa has been reported to accumulate Se mainly in proteins. Thus the characterization of major Se-containing proteins is very important in the application of Se-rich silkworm pupas in food and drugs. In this study, crude proteins were extracted from Se-rich silkworm pupas, followed by DEAE-Sepharose and Sephedex G-75 chromatography. Se content was measured after each step to determine the highest Se-containing fraction for the next step of separation. The proteins obtained were analyzed using SDS-PAGE, followed by in-gel digestion with trypsin, and were characterized by MALDI-TOF MS and ESI-MS/MS. These data showed two proteins mainly accumulated Se in the silkworm pupas. Those two proteins were proven by mass spectrometry to be arylphorin and sex-specific storage-protein 2 precursor (SP-2), respectively. Both of them belong to the storage proteins of amino acids during metamorphosis and the non-feeding pupal stage. The results suggest that Se could be enriched by storage proteins and be supplied to silkworm pupas in accompany with amino acids for the synthesis of new Se-containing proteins and peptides.     

Electrophoretic mapping of highly homologous keratins: A novel marker peptide approach

Identification of the intermediate filament proteins (IFPs) in the wool proteome has formerly been hampered by limited sequence information, the high degree of IFP homology and their close proximity on 2‐DE maps. This has been partially rectified by the recent acquisition of four new Type I and two Type II wool IFP sequences. Among closely migrating proteins, such as IFP clusters in a 2‐DE map, proteins with higher sequence coverage will be assigned higher scores, but the identification of unique peptides in such tight clusters may distinguish these closely migrating proteins. Two approaches were adopted for the study of wool IFPs. In the first, searches were conducted for peptides known to be unique to each member of the family in each spot. In the second, MALDI imaging was employed to examine peptides bound to a PVDF membrane from a poorly resolved part of the Type I IFP region of the 2‐DE map. As a result, a distinct picture has emerged of the distribution of the six Type I and four Type II IFPs across the 2‐DE wool protein map.     

一种新的人血浆中含硒蛋白测定方法

提出一个综合聚丙酰胺凝胶电泳分离和２,３－二氨基萘柱前衍生正相液体色谱测定硒的方法（ＤＡＮ－ＨＰＬＣ－ＦＬＤ）,分离测定了高硒地区人血浆中的含硒蛋白。在所鉴定的５种含硒蛋白中,其中至少有３种是从未明确鉴定过的含硒蛋白。     

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two‐dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI‐MS, LC‐Q‐TOF MS and LC‐Orbitrap Velos MS for the identification of proteins within one spot. With LC‐Orbitrap Velos MS each Coomassie Blue‐stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large‐scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low‐abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE‐MS to separate at the protein species level. Therefore, 2DE coupled with high‐sensitivity LC‐MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom‐up LC‐MS investigations.     

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC‐MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an “overlap score,” (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 “overlap factors,” (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.     

Selenium research in mammals using nuclear analytical methods and related techniques in conjunction with biochemical procedures

Due to the essential functions of selenium-containing enzymes and the relationships between changes in the selenium status and diseases, the determination of the element and its compounds is of great interest. Radiotracer studies with ⁷⁵Se have been valuable tools in selenium research. NAA and ICP-MS allow both total element and stable isotope measurements. ICP-MS in conjunction with chromatographic separation techniques and gel electrophoretic procedures coupled with scanning methods such as XRF, PIXE and laser ablation ICP-MS have been used in the determination of the selenium compounds. In this survey the application of these methods in selenium research is discussed with the help of examples on the regulation of the selenium metabolism and the detection and investigation of novel selenium-containing proteins. This revised version was published online in August 2006 with corrections to the Cover Date.     

Alternative sample preparation prior to two-dimensional electrophoresis protein analysis on solid lipid nanoparticles

The proteins adsorbing onto the surface of intravenously injected drug carriers are regarded as a key factor determining the organ distribution. Depending on the particle surface properties, certain proteins will be preferentially adsorbed, leading to the adherence of the particle to cells with the appropriate receptor. Therefore, the knowledge of the protein adsorption pattern and the correlation to in vivo behavior opens the perspective for the development of intravenous colloidal carriers for drug targeting. After incubation in plasma, the adsorbed proteins were analyzed using two-dimensional polyacrylamide gel electrophoresesis (2-D PAGE, 2-DE). The purpose of the present study was to develop an alternative separation method to separate solid lipid nanoparticles (SLN) carriers from plasma by gel filtration prior to 2-D PAGE. Via the specific absorption coefficients and a two-equation system, elution fractions were identified being practically plasma-free. This allows protein analysis on SLN which are typically in density too close to the density value of water to be separated by the standard centrifugation method. The SLN used for establishing the gel filtration were prepared in a way that they had a sufficiently low density to be additionally separated by centrifugation. The adsorption patterns obtained after separation with both methods were qualitatively and quantitatively identical, showing the suitability of the gel filtration.     

A metallomics approach discovers selenium-containing proteins in selenium-enriched soybean

Our previous study found that high-molecular-weight selenium (Se) species make up 82% of the total Se in the bean of Se-enriched soybean plants (Chan et al. 2010, Metallomics, 2(2): p. 147–153). The Se species have been commonly seen in other plants in addition to soybean, but their identities remain unresolved. The present study employs a multi-technique metallomics approach to characterize the proteins containing Se in the beans of Se-enriched soybean plants. Two main categories of proteins, maturation proteins and protease inhibitors, were found in Se-containing high-performance liquid chromatography (HPLC) fractions. The proteins were screened by two-dimensional HPLC-inductively coupled plasma mass spectrometry, size-exclusion chromatography, and anion-exchange chromatography, and the Se-containing fractions were then identified by peptide mapping using HPLC-Chip-electrospray ion trap mass spectrometry. Based on the belief that Se goes into proteins through non-specific incorporation, a new method was designed and applied for the Se-containing peptide identification. The Se-containing peptide KSDQSSSYDDDEYSKPCCDLCMCTRS, part of the sequence of protein Bowman–Birk proteinase isoinhibitor (Glycine max), was found in one of the Se-containing fractions. The nutritional value of the Se-containing proteins in Se-enriched soybeans will be an interesting topic for the future studies.     

Improved resolution in the acidic and basic region of 2‐DE of insect antennae proteins using hydroxyethyl disulfide

Olfaction is essential for processing chemical signals in insects, but characterizing the proteins implicated in this process has proved challenging. We optimized 2‐DE gel resolution of insect proteins by using a buffer containing two reducing agents, DTT and hydroxyethyl disulfide. This buffer clearly improved resolution and decreased spot streaking and spot trains of 2‐DE in comparison to DTT alone. We described for the first time that the buffer with DTT and hydroxyethyl disulfide further to reducing streaking in the basic part of the gel eliminates false spots in the acidic gel regions that appeared when only DTT was used as reducing agent.     

Comparative fluorescence two-dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on-gel generation of a reference protein spot grid

The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories.     

Anisotropic charge carrier transport of optoelectronic functional selenium-containing organic semiconductor materials

Organic semiconductors (OSCs) materials are currently under intense investigation because of their potential applications such as organic field-effect transistors, organic photovoltaic devices, and organic light-emitting diodes. Inspired by the selenization strategy can promote anisotropic charge carrier migration, and selenium-containing compounds have been proved to be promising materials as OSCs both for hole and electron transfer. Herein, we now explore the anisotropic transport properties of the series of selenium-containing compounds. For the compound containing Se Se bond, the Se Se bond will break when attaching an electron, thus those compounds cannot act as n-type OSCs. About the different isomer compounds with conjugated structure, the charge transfer will be affected by the stacking of the conjugated structures. The analysis of chemical structure and charge transfer property indicates that Se-containing materials are promising high-performance OSCs and might be used as p-type, n-type, or ambipolar OSCs. Furthermore, the symmetry of the selenium-containing OSCs will affect the type of OSCs. In addition, there is no direct relationship between the R groups with their performance, whether it or not as p-type OSCs or n-types. This work demonstrates the relationship between the optoelectronic function and structure of selenium-containing OSCs materials and hence paves the way to design and improve optoelectronic function of OSCs materials.     

Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999).

By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).