Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.     

Determination and distribution of human plasma selenoproteins

Major portions of plasma-selenium are incorporated in the proteins glutathione peroxidase (GSH-Px), selenoprotein P (Sel P) and albumin. A chromatographic method, adapted from a procedure by Harrison et al. [6], uses heparin- and blue-sepharose to separate the three protein fractions. The determination of selenium was carried out by electrothermal atomic absorption spectroscopy (ETAAS) using the Zeeman effect. The selenium distribution of 17 healthy subjects was 68 ± 7% of the total plasma selenium associated to Sel P, 25 ± 4% associated to p-GSH-Px and 7±4% associated to albumin. The recovery of selenium was 99 ± 4%. For precision measurements a plasma pool has been separated seven times. The selectivity of this method was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and GSH-Px activity measurements. A fast method, adapted for clinical applications, is described which allows to determine the human plasma selenium distribution in about an hour.     

Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorption/ionization.

The molecular masses of electroblotted proteins were determined with a time-of-flight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.     

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.     

Solar blind photocell – a simple element specific detector in Hg, As and Se speciation

An element-specific detection method, based on atomic absorption spectrometry (AAS) using solar blind photocells instead of a dispersion system, is described for the determination of Hg-, As-, and Se-species. Spectrometric investigations of AAS background lamps for As and Se measured with a CsI-cathode photocell shows its quality as narrow band detector. Species determination can be carried out subsequently to prior separation by HPLC or GC. The LODs for alkylated Hg species were below 1 ng/L, and for methylated As species below 1 μg/L. The relative standard deviation was < 10%. With the components described the production of cheap and automated dedicated speciation spectrometers is possible. Received: 18 May 1999 / Revised: 6 September 1999 / Accepted: 13 September 1999     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.     

Characterization of sodium dodecylsulfate polyacrylamide gel electrophoresis-separated M. agalactiae membrane antigens by mass spectrometry

Mycoplasma membrane proteins are generally designated according to their apparent molecular weight measured by SDS-PAGE. Several results about mycoplasma membrane antigens are conflicting because some doubts are emerging about the accuracy of the method utilised to identify the antigens. Aim of this work, was to characterise proteins separated after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)-mass spectrometry to allow an uncontroversial designation of the antigens. Fifteen proteins with molecular weights ranging from 15,000 to 80,000 Da had been excised from gel and their whole molecular weight and proteolytic pattern had been determined using MALDI-TOF. The peptide pattern obtained using trypsin digestion allowed us to identify LipA, P48, P59, P80 and P40. Some other proteins showed analogies to proteins of Mycoplasma genitalium or Mycoplasma pneumoniae the only Mycoplasmas completely sequenced. There wasn't a close correspondence between the SDS-PAGE apparent molecular weight (generally used to name the proteins), the gene derived calculated mass and the molecular weight of whole proteins measured by MALDI-TOF. Only micro sequence data obtained by MS/MS allowed us to identify LipC, described as one of the most important Mycoplasma agalactiae antigens. This protein was found in correspondence with the 50 kDa region, instead of the 25 kDa region, confirming a phenomenon that we previously described.     

Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

Separation and detection of selenium-containing proteins in human serum

Selenium-containing proteins or their subunits in human serum were separated and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the amount of selenium in each protein band was determined by HPLC with a fluorescence detector after derivatization with 2,4-diaminonaphthalene (DAN). This procedure provides a detection limit of 0.06 ng in a linear range of 0–1.5 ng. A protein is defined as a selenium-containing protein if its mean Se content exceeds twice the detection limit (0.12 ng) and twice the standard deviation of three replicates in sample determination. At least 4 selenium-containing bands with apparent molecular masses of 57–74, 46–56, 40–42 and 21–22 kDa could be detected from human serum collected from 4 volunteers.     

Separation and detection of selenium-containing proteins in human serum

Selenium-containing proteins or their subunits in human serum were separated and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the amount of selenium in each protein band was determined by HPLC with a fluorescence detector after derivatization with 2,4-diaminonaphthalene (DAN). This procedure provides a detection limit of 0.06 ng in a linear range of 0-1.5 ng. A protein is defined as a selenium-containing protein if its mean Se content exceeds twice the detection limit (0.12 ng) and twice the standard deviation of three replicates in sample determination. At least 4 selenium-containing bands with apparent molecular masses of 57-74, 46-56, 40-42 and 21-22 kDa could be detected from human serum collected from 4 volunteers.     

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 platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins

Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine-phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and phosphorylated proteins were detected by Western blotting using anti-phosphotyrosine antibodies. Visualized protein spots were excised, digested with trypsin and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS-Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti-phosphotyrosine antibody. A method for immunoprecipitation of tyrosine-phosphorylated peptides was optimized.     

Selenoproteins: the key factor in selenium essentiality. State of the art analytical techniques for selenoprotein studies

Selenium is an essential element for human health. The benefits of selenium are many including protection against cancer, heart diseases and other cardiovascular and muscle disorders. Selenium is also helpful in controlling gastrointestinal disorders, enhancing immunity of the human body and reducing age-related diseases. The health-promoting properties of Se are due to vital functions of selenoproteins in which selenium is present as selenocysteine, the 21st amino acid. To date, dozens of selenoprotein families have been described though many have roles that have not been fully elucidated. Selenoproteins research has attracted tremendous interest from different scientific areas. Analytical chemists have not remained indifferent to the attractive features of these unique proteins. Different analytical techniques, such as multidimensional chromatography–inductively coupled plasma mass spectrometry (ICPMS), electrospray (tandem) mass spectrometry (ESI-MS/MS), matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis–laser ablation inductively coupled plasma mass spectrometry (SDS-PAGE-LA-ICPMS), have been applied to the determination of selenoproteins and selenium-containing proteins. This review describes the best-characterized selenoproteins to date in addition to the major contributions of analytical chemistry to the field of selenoproteins. The article also highlights the challenges of combining elemental and molecular mass spectrometry for the determination of selenoproteins and selenium-containing proteins.     

PHOTOCHEMICAL BINDING OF PHENOTHIAZINES ON BIOLOGICAL MEMBRANE PROTEINS

Abstract— The photobinding of phenothiazine derivatives (chlorpromazine, fluphenazine, promazine and promethazine) was studied on four different types of biological membranes (microsomes, myelin and synaptosomes from rat brain as well as human erythrocytes). The photoreaction was performed by ultraviolet irradiation of the tritiated compounds in their long wavelength absorption band (313 nm) and bound photoproducts were analysed by autoradiography of the proteins separated by polyacrylamide gel electrophoresis. The specificity of binding is low, however, a 34000 dalton band is intensely labeled on synaptic membranes with chlorpromazine and fluphenazine. All the phenothiazines bind on erythrocyte membrane proteins and specially on band 4.2 and on a peptide located before actin on the electrophoresis gel. These results show the generality of the phenothiazine photobinding on membrane proteins. These photobinding properties can be used for the identification and localization of some of these proteins.     

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.