Comparison of TCA and collagenase in the isolation of tissue collagen.

A rapid method of peptide mapping by thin layer electrophoresis and chromatography is described that permits good maps to be produced from as little as 0.5 nmole of protein by means of conventional staining procedures with ninhydrin. The method can be made even more sensitive by prior amidination of the protein amino groups using methyl-[1-¹⁴C]acetimidate, a simple synthesis of which is given. Because proteins generally contain many amino groups, autoradiographs of peptide maps of such radiolabeled proteins show a large number of spots in characteristic patterns, and the derivatization of the protein is easy and specific. A cheap and simply constructed apparatus for carrying out the thin layer electrophoresis is also described.     

Assay of proteins by Lowry's method in the presence of high concentrations of β-mercaptoethanol

β-Mercaptoethanol interferes in the determination of protein by the Lowry method (1–6). The interference can be overcome by the precipitation of proteins by trichloroacetic acid or acetone or by the use of H₂O₂ which oxidizes the sulfhydryl groups of β-mercaptoethanol (5). Both these methods have inherent disadvantages. Ross and Schatz (5) described a procedure for protein determination in the presence of high concentrations of β-mercaptoethanol where they removed the interference by the addition of iodoacetate. But addition of iodoacetate decreased the sensitivity of the reaction. The removal of interference by β-mercaptoethanol by heating has also been reported (3), but we observed that this procedure is not feasible when a large amount of β-mercaptoethanol is present in the protein samples. In the method reported in this communication, we made use of vacuum drying for the removal of interference by β-merceptoethanol. This method is simple, sensitive, takes less time, and can be used for the determination of protein in the presence of β-mercaptoethanol at levels as high as 10% in a sample volume of 1.0 ml (1.43 mmol) without using any additional chemical steps.     

Low molecular weight silk proteins in Galleria mellonella

Galleria cocoon proteins have been extracted by different solubilizing agents. Nine protein bands were observed by gel electrophoresis, with molecular weights ranging from 18 to 420 kD. Three silk proteins of 24, 29 and 30 kD were extracted only in the presence of β-mercaptoethanol, suggesting that they are covalently linked by disulfide bonds to the large fibroin. They are likely to be the products of the highly abundant mRNA of the posterior silk gland cells. In vitro translation analysis of this mRNA yielded 24, 29 and 30 kD proteins. Thus, as in Bombyx, the Galleria silk is composed of several subunits, including fibroin and low molecular weight polypeptides. However, the genes coding for fibroin or low molecular weight silk proteins in Bombyx and Galleria do not show nucleotide base homology.     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

A new method for partial peptide mapping using N-chlorosuccinimide/urea and peptide silver staining in sodium dodecyl sulfate-polyacrylamide gels

A simple, rapid procedure for obtaining partial peptide maps from nanogram quantities of protein in gel slices using the selective tryptophanyl peptide bond cleavage reagent N-chlorosuccinimide/urea is presented. The generated peptide fragments can be visualized by autoradiography or by a sensitive protein silver-staining technique.     

Isolation and characterization of tracheobronchial mucin from a laryngectomee

A tracheobronchial mucin was isolated from the tracheobronchial secretion of a laryngectomee. It was purified by gel filtration on Sepharose CL-6B in Trisurea buffer and rechromatography of excluded materials through the same gel matrix. It was homogeneous in 0.7% agarose-2% polyacrylamide electrophoresis under nonreducing conditions. Comparable analysis with 2-mercaptoethanol revealed at least 3 subunits. Based upon recoverable weight, the mucin was composed of 75% carbohydrate, 21% protein, and 3% sulfate. Oligosaccharides obtained by alkaline β-elimination indicated O-glycosyl linkage to the peptide component. Marked heterogeneity of the carbohydrate side-chains was reflected in the preparation of 20 distinct oligosaccharides ranging in size from 4 to 17 residues.     

Changes in the sarcoplasmic reticulum ATPase protein under denaturing conditions used for sodium dodecyl sulfate--polyacrylamide gel electrophoresis

The electrophoretic pattern of the sarcoplasmic reticulum (SR) ATPase protein was found to change, depending on the conditions used to denature the SR vesicles in sodium dodecyl sulfate (SDS), prior to SDS-polyacrylamide gel electrophoresis. A smearing of the gel pattern above the ATPase protein was observed when the SR vesicles were denatured at 100 °C in SDS, in the absence of β-mercaptoethanol (β-ME). Denaturation of the SR vesicles in SDS at 100 °C in the presence and the absence of β-ME reduced the amount of SR ATPase protein by half. More high-molecular-weight aggregates were formed in the presence than in the absence of β-ME. The other proteins of the SR as well as myofibrils and bovine serum albumin were found to be relatively unaffected by these treatments. It is concluded that, for the study of SR ATPase protein by SDS-polyacrylamide gel electrophoresis, denaturation at 100 °C should be avoided.     

Anticancer compound Oplopantriol A kills cancer cells through inducing ER stress and BH3 proteins Bim and Noxa

Oplopantriol-A (OPT) is a natural polyyne from Oplopanax horridus. We show here that OPT preferentially kills cancer cells and inhibits tumor growth. We demonstrate that OPT-induced cancer cell death is mediated by excessive endoplasmic reticulum (ER) stress. Decreasing the level of ER stress either by inactivating components of the unfolded protein response (UPR) pathway or by expression of ER chaperone protein glucose-regulated protein 78 (GRP78) decreases OPT-induced cell death. We show that OPT induces the accumulation of ubiquitinated proteins and the stabilization of unstable proteins, suggesting that OPT functions, at least in part, through interfering with the ubiquitin/proteasome pathway. In support of this, inhibition of protein synthesis significantly decreased the accumulation of ubiquitinated proteins, which is correlated with significantly decreased OPT-induced ER stress and cell death. Finally, we show that OPT treatment significantly induced the expression of BH3-only proteins, Noxa and Bim. Knockdown of both Noxa and Bim significantly blocked OPT-induced cell death. Taken together, our results suggest that OPT is a potential new anticancer agent that induces cancer cell death through inducing ER stress and BH3 proteins Noxa and Bim.     

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (>500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoresic dye front. Inclusion of 10 mm 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mm 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

Scanning of polyacrylamide gel electrophoresis columns for detection of unstained protein zones and for their localization relative to enzyme activities.

Background disturbances which often confuse 280-nm scans of polyacrylamide gels, can be distinguished from true protein peaks by scanning also at a wavelength where the proteins do not absorb (for instance 310 nm).A scanning technique has been used also for precise localization of spectrophotometrically detectable enzyme activities relative to protein zones. After electrophoresis the gel is transferred to a specially designed quartz cuvette and scanned at 280 nm for protein detection. The substrate is then allowed to diffuse into the gel and the activity is located by scanning at a wavelength absorbed by the product.Scanning of polyacrylamide gel electrophoresis columns can be used for the study of solute-solute interactions, as illustrated by a simple model experiment on the binding of bilirubin to albumin.     

Author index for volume 84

A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of α- and β-chains were mixed with 300× molar excess of β-mercaptoethanol over the p-hydroxy mercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mm phosphate buffer-1 mm Na₂EDTA-47 mm β-mercaptoethanol, pH 5.85 and then with 10 mm phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mm K₂HPO₄. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.     

A rapid percoll gradient procedure for the preparation of acetylcholine receptor-rich vesicles from Torpedo marmorata electric organ

A rapid method for the isolation of acetylcholine receptor-rich membranes from Torpedo marmorata electric organ, using a Percoll density gradient, is presented. The preparation of purified membranes appeared on electron microscope examination as a homogeneous population of sealed vesicles, covered with the characteristic rosettes identified as acetylcholine receptor clusters. Biochemical characterization revealed an α-bungarotoxin specific binding activity of 1.6–2.1 nmol/mg of protein, low acetylcholinesterase specific activity, a protein:lipid ratio (w/w) of 2.1 with high cholesterol content. Polyacrylamide gel electrophoresis under denaturing conditions showed the polypeptide bands characteristic of the receptor (α, β, γ and δ), together with 43 kDa and ～100 kDa proteins (already described as ν and ?).The method is simple and rapid, and maintains constant osmotic conditions throughout. It thus represents an improvement over previous methods, and will be useful for routine preparation and specially for studying post-synaptic membrane components interactions.     

Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae

The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses

Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information.     

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.     

A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances

A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.     

Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents

We encountered β-mercaptoethanol-dependent artifact signals in western blot analyses using polyclonal antisera. Replacing β-mercaptoethanol with dithiothreitol in the loading buffer did not eliminate the artifact signals. However, lowering the concentration of either dithiothreitol or β-mercaptoethanol eliminated the background problems and allowed specific detection of the target protein. These results are consistent with the background signal being caused by anti-keratin antibodies in the antisera and keratin contamination of reagents. This study highlights the importance of testing a range of reducing agent concentrations when trying to eliminate artifact bands from western blots. However, this method may not be applicable when target proteins have disulfide bridges.     

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.