Extraction and characterization of adenovirus

A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.     

Applications of an automated apparatus for two-dimensional electrophoresis, Model TEP-1, for microsequence analyses of proteins.

Two-dimensional electrophoresis has become a rapid and powerful separation method for the preparation of samples for sequence analysis depending on the development of automated microsequencers accompanied by a blotting technique. We have constructed an automated apparatus for two-dimensional electrophoresis, Model TEP-1, which allows precise analytical runs under computer-control with a fully automated transfer from the first- to the second-dimensional gel. In the present paper we describe several applications of TEP-1 followed by blotting and sequencing to reveal the primary structure of proteins in a complex mixture. Several known and unknown proteins were analyzed. Enzymatic digests of proteins could be directly applied onto the TEP-1, in which a gel of lower molecular weight proteins was used as the second dimension. Subsequent blotting and sequencing provided internal sequence information. The use of TEP-1, followed by blotting, provides spots containing ca. 10-100 pmol protein. These figures permitted the identification of 10-40 amino acid residues from the N-terminus. The TEP-1 provides a simple and rapid separation of complex mixtures obtained from natural sources and chemical, biological or genetic products.     

Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products

Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes‐AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes‐AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA‐ICP‐MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs‐protein binding (what is not possible using SDS‐PAGE).     

On-line capillary electrophoresis-mass spectrometry for the analysis of biomolecules

Mass spectrometry (MS) has become a key tool for the characterization of biologically relevant molecules in the last decade. Due to the complexity of most biological samples an upstream separation is essential. Capillary electrophoresis (CE) has gained much interest due to its high separation efficiency, speed, and often complementary selectivity to liquid chromatography. We describe the state-of-the-art of on-line CE-MS for the analysis of molecules of biological origin. The characterization of peptides, including the study of post-translational modifications, intact proteins, oligonucleotides, and related interaction studies are reviewed. Relevant publications are summarized in tables, including some important method parameters. Key applications are discussed with respect to the advantages and limitations of CE-MS. Coupling interfaces, preconcentration techniques, capillary coatings, and the different CE techniques, e.g., capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, etc. are briefly discussed against the background of their bioanalytical applications.     

Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance liquid chromatography. Whole lysates of cells and tissue were prefractionated by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electrophoresis (2-DE). The reproducibility of this prefractionation technique proved to be optimal for comparing 2-DE gels from two different cell states. In addition, this method is suitable for enriching low-abundance proteins barely detectable by silver staining to amounts that can be detected by Coomassie blue and further analyzed by mass spectrometry.     

Preparation of nitrilotriacetic acid/Co2+-linked, silica/boron-coated magnetite nanoparticles for purification of 6 x histidine-tagged proteins

In this report, we describe the preparation of novel nitrilotriacetic acid/Co2+-linked, silica/boron-coated magnetite nanoparticles for purification of 6 x His-tagged proteins. The nanoparticles were approximately 200 nm in size and were stable against hydrochloric acid and had negligible non-specific binding for protein. Elimination of non-specific binding by nucleic acids was readily achieved by digestion of samples with DNase and RNase. The modified nanoparticles were used to purify two model proteins: one had a C-terminal 6 x His tag, and the other had an internal 6 x His tag. Both proteins were purified within one hour into single band purity on sodium dodecyl sulfate-polyacrylamide electrophoresis gel.     

Study of Burkitt lymphoma cell line proteins by high resolution two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry

We paper describe a mass spectrometric approach generally applicable for the rapid identification and characterization of proteins isolated by two-dimensional gel electrophoresis (2-DE). The highly sensitive nanoflow-electrospray mass spectrometry employing a quadrupole-time of flight mass spectrometer was used for the direct identification of proteins from the peptide mixture generated from only one high resolution 2-DE gel without high performance liquid chromatography (HPLC) separation or Edman sequencing. Due to the high sensitivity and high mass accuracy of the instrument employed, this technique proved to be a powerful tool for the identification of proteins from femtomole amounts of materials. We applied the technique for the investigation of Burkitt lymphoma BL60 cell proteins. This cell line has been used as a model to assign apoptosis-associated proteins by subtractive analysis of normal and apoptotic cells. From the nuclear fraction of these cells, 36 protein spots were examined, from only one micropreparative Coomassie Brilliant Blue R-250 stained gel, after proteolytic digestion by matrix assisted laser desorption ionization (MALDI) and nanospray mass spectrometry (MS). In combination with database searches, of 33 proteins were successfully identified by nanospray-MS/MS-sequencing of up to eight peptides per protein. Three proteins were new proteins not listed in any of the available databases. Some of the identified proteins are known to be involved in apoptosis processes, the others were common proteins in the eukaryotic cell. The given technique and the protein data are the basis for construction of a database to compare normal and apoptosis-induced cells and, further, to enable fast screening of drug impact in apoptosis-associated processes.     

Electrophoretic separation of ubiquitin and single amino acid residue ubiquitin extensions using a commercial modified acrylamide gel electrophoresis system: an assay to determine catalytic capacities of deubiquitinating enzymes

We have chemically synthesised a number of ubiquitin extension proteins, with carboxyl-terminal single amino acid residue extensions, to use as substrates to assess the catalytic capacities of deubiquitinating enzymes (DUBs). Here we describe a modified acrylamide gel electrophoresis system which allows separation of peptide- or isopeptide-linked ubiquitin-lysine from ubiquitin (77 and 76 residue proteins respectively) in only 2 h. Western blotting, using antibodies against ubiquitin, allows both substrate (i.e. ubiquitin-lysine) and product (i.e. ubiquitin) of DUB-catalyzed cleavage reactions to be detected. Catalytic capacities of DUBs may be indicative of in vivo functions of these proteases.     

LC-nanospray-MS/MS analysis of hydrophobic proteins from membrane protein complexes isolated by blue-native electrophoresis

The application of two-dimensional electrophoresis for the identification of hydrophobic membrane proteins is principally hampered by precipitation of many of these proteins during first-dimension, isoelectric focusing. Therefore new strategies towards the identification and characterization of membrane proteins are being developed. In this work we present a direct and rapid approach from blue-native gels to mass spectrometry, which allows the analyses of complete complexes and prevents protein aggregation of hydrophobic regions during electrophoresis. We combine blue-native gel electrophoresis and liquid chromatography--nanospray-iontrap tandem mass spectrometry to analyze the composition of oxidative phosphorylation complexes I, III, IV and V from bovine-heart mitochondria as a model system containing a number of highly hydrophobic proteins. Bands from blue-native gels were subjected either to in-gel or to in-solution tryptic digestion. The obtained peptide mixtures were further analyzed by liquid chromatography--tandem mass spectrometry and the corresponding proteins were identified by database search. From a total of 86 proteins, 67 protein subunits could be identified including all highly hydrophobic components, except the ND4L and ND6 subunits of complex I. We demonstrate that liquid chromatography--tandem mass spectrometry combined to blue-native electrophoresis is a straightforward tool for proteomic analysis of multiprotein complexes, and especially for the identification of very hydrophobic membrane protein constituents that are not accessible by common isoelectric focusing/sodium dodecyl sulphate gel electrophoresis.     

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis application to cerebellar protein mapping

A method is presented for the structural characterization of proteins separated by two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 × 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.     

Characterization of ribosomal proteins as biomarkers for matrix-assisted laser desorption/ionization mass spectral identification of Lactobacillus plantarum

For rapid identification of bacteria by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), a bioinformatics approach using ribosomal subunit proteins as biomarkers has been proposed. This method compares the observed masses for biomarkers with calculated masses as predicted from the amino acid sequences registered on protein databases. To evaluate this approach, the expressed ribosomal proteins of a genome-sequenced bacterium, Lactobacillus plantarum NCIMB 8826, were characterized as a model sample. The protein expression of 42 ribosomal subunit proteins, together with 10 ribosome-associated proteins in the isolated ribosome fraction, was confirmed through two-dimensional gel electrophoresis combined with peptide mass fingerprinting. The observed masses of the proteins in the isolated ribosome fraction were then determined by MALDI-MS. We preliminarily selected 44 biomarkers whose observed masses were matched with the calculated masses predicted from the amino acid sequence registered in the protein databases by considering N-terminal methionine loss only. Of these, the finally selected reliable biomarkers were 34 proteins including 31 ribosomal subunit proteins and 3 ribosome-associated proteins that could be observed in the MALDI mass spectra of the cell lysate sample. These biomarkers were usable in MALDI-MS characterization of two industrial L. plantarum cultures.     

Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources

Purification and chemical characterization of proteins may be achieved by combining two-dimensional electrophoresis (2-DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2-DE pattern as possible. Under optimized conditions, blotting efficiencies between 60% and 100% were obtained for five marker proteins; the mean values from four regions of a 2-DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60% and 79%. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patterns, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.     

Identification of a chemical substructure that is immobilized to ferrite nanoparticles (FP)

Despite the wide utility of ferrite nanoparticles (FP), a methodology to conjugate heterologous molecules to FP is still limited and characterization of small molecule-conjugated FP is not well known. Here, we describe what kinds of proteins and amino acids are selectively immobilized onto FP when FP is synthesized in the presence of these molecules. Two-dimentional gel electrophoresis (2D SDS-PAGE) showed that proteins with low pI value were selectively bound to FP. Quantitative analyses using HPLC suggested that L-aspartic acid (Asp) and L-cysteine (Cys) were bound to FP selectively among natural amino acids examined. Additional analysis of compounds-conjugated FP revealed that selective binding of Asp to FP was attributed with its molecular structure. It was found that the substructure of amino acid-bound to FP specifically was composed of a defined chelation of two carboxyl groups separated by two carbon atoms as deduced from FT-IR measurement. Thus, we concluded that molecules possessing two carboxyl groups separated by two carbons were bound to FP spontaneously and selectively, which might enable the attachment of free functional groups onto the FP surface if their molecules have functional groups other than carboxyl groups. The resulting complex might be applicable as a chemical tag to immobilize various molecules onto FP.     

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.     

Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

High-throughput DNA sequencing has resulted in increasing input in protein sequence databases. Today more than 20 genomes have been sequenced and many more will be completed in the near future, including the largest of them all, the human genome. Presently, sequence databases contain entries for more than 425.000 protein sequences. However, the cellular functions are determined by the set of proteins expressed in the cell--the proteome. Two-dimensional gel electrophoresis, mass spectrometry and bioinformatics have become important tools in correlating the proteome with the genome. The current dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated.     

Identification of the apolipoprotein E4 isoform in cerebrospinal fluid with preparative two-dimensional electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

Apolipoprotein E (apoE) was isolated from human cerebrospinal fluid (CSF) from control individuals and patients with Alzheimer's disease (AD). The purification was performed with preparative two-dimensional electrophoresis (2-DE), involving liquid-phase isoelectric focusing (IEF) in the Rotofor cell in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution in the Mini Whole Gel Eluter. ApoE was characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of tryptic digests. The known change of Cys to Arg in position 112 of the apoE4 isoform was identified. This was detected in CSF from AD patients, reflecting the increased frequency of the apoE4 allele in this population. This peptide was not detected in CSF samples from healty control individuals. The use of this rapid electrophoretic separation in proteomic studies of CSF proteins provides single proteins, such as apoE, of high purity in yields sufficient for characterization by MALDI-TOF-MS. Characterization of proteins and their modifications (amino acid substitutions, glycosylation or phosphorylation) in CSF will be a useful tool in the investigation of the pathophysiology of brain disorders such as AD.     

Proteomic Evaluation of Biomarkers to Determine the Postmortem Interval

Proteomics separates and analyzes proteins for investigation at the cellular level in regard to disease processing by analyzing the proteins’ expression, function, structure, post-translational modification, and protein–protein interaction. In general forensic investigations, the postmortem interval was evaluated by measuring changes in body temperature after death, along with forensic entomological knowledge. These investigations may be restrictive and subjective because of external factors. The objectives of this study are to sort biomarker candidates and develop a direct postmortem-interval characterization method using proteomics through analyzing and tracking down proteins in the deceased that change in accordance with the postmortem interval. The liver and heart tissues of rats were collected for protein extraction in the 24-h interval following death, and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis was conducted based on the isoelectric point and the molecular weight for separation. To validate protein spot changes on the gel, the stained electrophoresis gels were scanned and converted to digital images. Through image analysis, 14 liver proteins and 12 heart proteins were sorted and classified into four groups based on pattern changes. These proteins containing spots were extracted from the gel and analyzed by high-performance liquid chromatography with tandem mass spectrometry. Finally, 26 protein postmortem-interval relevant biomarker candidates were identified using software. Some of the proteins were muscle proteins while others were oxidation-related proteins. This study presents a new approach to the postmortem-interval research using proteomics and could be substituted for postmortem-interval evaluation.     

Improving the detection of proteins after transfer to polyvinylidene difluoride membranes.

N-Terminal sequence analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes has become the method for molecular characterization of proteins contained in biological samples. However, the proteins of lower abundance cannot be sequenced directly, without improving the technique. We have studied a drying method on several PVDF membranes including Trans-Blott, Immobilon P and Problott. Using Amido Black, Coomassie Brilliant Blue R-250 and Ponceau S, we have obtained, in comparison with the non-dried membranes, an enormous increase in the number of detectable proteins.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

A simple system for staining protein and nucleic acid electrophoresis gels

Researchers in molecular biology spend a significant amount of time tending to the staining and destaining of electrophoresis gels. Here we describe a simple system, costing approximately $100 and taking approximately 1 h to assemble, that automates standard nucleic acid and protein gel staining protocols. Staining is done in a tray or, with DNA gels, in the electrophoresis chamber itself following automatic detection of the voltage drop. Miniature pumps controlled by a microcontroller chip exchange the necessary solutions at programmed time intervals. We demonstrate efficient and highly reproducible ethidium bromide and methylene blue staining of DNA in agarose gels and Coomassie blue and silver staining of proteins in polyacrylamide gels.