Showing papers in "Electrophoresis in 1998"
TL;DR: Quantitative analysis of protein expression data obtained by high‐throughput methods has led to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo.
Abstract: The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.
1,005 citations
TL;DR: It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.
Abstract: Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.
766 citations
TL;DR: The extraction and enrichment of membrane proteins for separation by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) after differential solubilization of an Escherichia coli cell lysate is described and 11 membrane proteins are identified from this pellet.
Abstract: We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.
479 citations
TL;DR: It is shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing, and modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two‐dimensional gels.
Abstract: The separation of membrane proteins by high-resolution two-dimensional electrophoresis was carried out. At high loads, these proteins are prone to precipitation, resulting in poor resolution. It is shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing. As thiourea inhibits acrylamide polymerization, a modified photopolymerization system must be used. These modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two-dimensional gels.
335 citations
TL;DR: Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14–16 carbons proved most efficient, solubilizing previously undetected membrane proteins, improving the solubility of proteins in this technique.
Abstract: Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14–16 carbons proved most efficient, solubilizing previously undetected membrane proteins.
332 citations
TL;DR: Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution, and its use in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step.
Abstract: In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first-dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second-dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol-specific alkylating reagents.
239 citations
TL;DR: Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases, including databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences.
Abstract: Large-scale DNA sequencing is creating a sequence infrastructure of great benefit to protein biochemistry. Concurrent with the application of large-scale DNA sequencing to whole genome analysis, mass spectrometry has attained the capability to rapidly, and with remarkable sensitivity, determine weights and amino acid sequences of peptides. Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases. When a protein is digested with a site-specific protease, the molecular weights of the resulting collection of peptides, the mass map or fingerprint, can be determined using mass spectrometry. The molecular weights of the set of peptides derived from the digestion of a protein can then be used to identify the protein. Several different approaches have been developed. Protein identification using peptide mass mapping is an effective technique when studying organisms with completed genomes. A second method is based on the use of data created by tandem mass spectrometers. Tandem mass spectra contain highly specific information in the fragmentation pattern as well as sequence information. This information has been used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences. The ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but a large amount of expressed gene sequence is available (e.g., human and mouse). Furthermore, a strength of using tandem mass spectra to search databases is the ability to identify proteins present in fairly complex mixtures.
233 citations
TL;DR: It is concluded that proteome analysis is an essential tool in the understanding of regulated biological systems and will continue to be enhanced by further improvements in analytical technology.
Abstract: In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.
220 citations
TL;DR: A software tool, PepFrag, for searching protein and DNA sequence databases that can use different types of mass spectrometric information to restrict the search is described.
Abstract: In an effort to gain an understanding of the value of the information in different mass spectrometric measurements for protein identification, the genome of Saccharomyces cerevisiae was studied in silico. We calculate how constraining the knowledge of the mass of a proteolytic peptide is as a function of mass and mass accuracy. We also assess the value for protein identification of additional information concerning a proteolytic peptide, including the presence or absence of a given amino acid, the number of exchangeable hydrogens, the N-terminal sequence, and the masses of mass spectrometrically produced fragment ions. Knowledge of the relative value of these different constraints is useful in the design of efficient protein identification experiments. Finally, we describe a software tool, PepFrag, for searching protein and DNA sequence databases that can use different types of mass spectrometric information to restrict the search.
202 citations
TL;DR: The construction of the human mitochondrial proteome is reported, using placenta as the source tissue and the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome.
Abstract: Owing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and “physiology” of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N-terminal microsequencing, blotting). The optimization steps in two-dimensional (2-D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies1 .
195 citations
TL;DR: Investigation of the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two‐dimensional gel concluded that further developments of 2‐D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.
Abstract: Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel. Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2-D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.
TL;DR: In this article, a matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots.
Abstract: Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest.
TL;DR: This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D‐Ala‐D‐ Ala (DADA)‐based peptidyl ligands, and variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution.
Abstract: Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D-Ala-D-Ala (DADA)-based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins - protein charge ladders - and ACE is a physical-organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N-terminal amino group.
TL;DR: These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas.
Abstract: This review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electroosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.
TL;DR: The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA.
Abstract: Two-dimensional (2-D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin alpha1, haptoglobin alpha2, haptoglobin beta and alpha1-anti-chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin alpha2, haptoglobin beta, alpha1-antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A-I, apolipoprotein A-IV, transthyretin, Gc-globulin, alpha2-HS glycoprotein, alpha2-macroglobulin and alpha1-B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of alpha1-antiprotease and SAA but raised the levels of transthyretin, Gc-globulin, alpha2-HS-glycoprotein and alpha2-macroglobulin in RA patients. C-reactive protein (CRP) could not be quantified on 2-D gels but, when measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute-phase proteins in RA patients could clearly be distinguished from that of piroxicam.
TL;DR: It is found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide poreiameter distribution narrows as the gel concentration increases.
Abstract: Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.
TL;DR: The two major services the resource laboratory employs when providing protein identification from in‐gel or PVDF‐bound proteins are described.
Abstract: As the resource laboratory for Rockefeller University our emphasis continues to be on methodology development for the routine analysis of low abundance proteins isolated from native sources. In the past ten years, gel electrophoresis of proteins has become the method of choice for the preparation of microgram and submicrogram quantities of protein for primary structural characterization, and over 95% of the samples submitted for protein identification are either in a gel or bound to polyvinyl difluoride membranes (PVDF). As such, we employ multiple microanalytical approaches encompassing Edman sequence degradation, amino acid and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis to provide an integrated protein characterization of such samples. Here we describe the two major services we employ when providing protein identification from in-gel or PVDF-bound proteins.
TL;DR: A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE) and gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing.
Abstract: A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing in 50 microm capillary at 42 degrees C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA-capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high-throughput instruments for DNA sequencing.
TL;DR: It is concluded that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100.
Abstract: The analysis of the fine structure of starches is important to the investigation of linkages between starch structure and function and to the investigation of the properties and roles of starch biosynthetic, modifying and degradation enzymes. Fluorophore-assisted carbohydrate electrophoresis has recently been introduced as a method for the analysis of the oligosaccharide populations released by the enzymatic digestion of starches, which has advantages in resolution and sensitivity over previously used methods, and provides the capacity for the facile analysis of oligosaccharide populations on either a molar or mass basis. The use of fluorophore-assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of labeling and separation techniques. Examples of separations using slab gel electrophoresis, DNA sequencer analysis and capillary electrophoresis are presented and we conclude that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100. Examples of isoamylase-debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through the analysis of oligosaccharides released by the debranching of limit dextrins derived from starches and glycogens is introduced as a useful diagnostic of starch structure. The potential for future development of novel diagnostics for starch structure using fluorophore-assisted carbohydrate electrophoresis is discussed.
TL;DR: This review evaluates the literature on continuous free flow electrophoresis, published during the last four years, and briefly describes the principles of the method and the techniques used, referring to fundamental papers published earlier.
Abstract: This review evaluates the literature on continuous free flow electrophoresis, published during the last four years. Its aim is to serve not only experts in the field but also newcomers, and, therefore, it also briefly describes the principles of the method and the techniques used, referring to fundamental papers published earlier. The actual commercial instrumentation is briefly outlined. A substantial part of this review is devoted to the optimization of the performance of this method. Finally, diverse applications of fractionations of charged species in solution, ranging from small ions to biological particles and cells, are surveyed.
TL;DR: The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelie ladder, allows for accurate genotypesing of STR loci.
Abstract: Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten flourescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler™ kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelie ladder, allows for accurate genotyping of STR loci. Sizing precision of ⩽ 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.
TL;DR: Significant signal suppression effects of ladder peptides were investigated using the 17‐amino acid peptide dynorphin A and a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
Abstract: The techniques of enzymatic and chemical peptide ladder sequencing, coupled with ultraviolet - matrix assisted laser desorption/ionization - mass spectrometry (UV-MALDI-MS) have been improving continuously in the last five years and have now become important tools for primary structure identification. In this work, signal suppression effects, appearing in UV-MALDI-MS (excitation 337 nm) of ladder peptides, were investigated using the 17-amino acid peptide dynorphin A. We show, with examples of simple "two-peptide" systems and more complex "multi-peptide" systems, that suppression effects do in fact exist. The magnitude of the observed suppression is strongly dependent upon both the nature and the amount of the suppressing peptide. Suppression behavior of individual ladder peptides was investigated on equimolar mixtures of ten ladder peptides. Significant signal suppression was recorded for all ladder peptides, with some of them being approximately 170 times lower in signal intensity than the pure, i.e., unsuppressed peptide at the same concentration. For the investigated system--dynorphin A, 4-hydroxy-alpha-cyanocinnamic acid (4-HCCA) matrix, UV excitation--a correlation between the extent of suppression and an intractable combination of peptide hydrophobicity and the presence of several basic amino acids can be seen.
TL;DR: The different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers, including charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids are presented.
Abstract: This review presents the different chiral selectors used in capillary electrophoresis (CE) for the separation of enantiomers. The use of charged cyclodextrins, crown ethers, polysaccharides, proteins, natural and synthetic micelles, macrocyclic antibiotics and ergot alkaloids is discussed in detail. Neutral native and derivatized cyclodextrins are not treated because several review articles have already been published on this topic. Recent developments like the application of two chiral selectors in the same background electrolyte are highlighted.
TL;DR: For checking theoretical two‐dimensional (2‐D) maps derived from sequenced genomes, indicating that nonnegligible amounts of proteins up to pH 12 are to be expected, a wide‐range immobilized pH 4–12 gradient was generated.
Abstract: For checking theoretical two-dimensional (2-D) maps derived from sequenced genomes, indicating that nonnegligible amounts of proteins up to pH 12 are to be expected, a wide-range immobilized pH 4-12 gradient was generated. Depending on the extraction method of sample preparation, proteins with pls up to pH 12 are detected in a single gel. Highly reproducible protein patterns focused to the steady state with round-shaped spots up to pH 12 are obtained with the standard protocol originally described in 1988 (Gorg et al., Electrophoresis 1988, 9, 531-546).
TL;DR: In this paper, an integrated injector, separator and post-separation reactor was fabricated on planar glass wafers, and the fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response.
Abstract: Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 microm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 microm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 microg/mL) with increasing mouse IgG (0-60 microg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were +/- 5.8 and +/- 1.3 %, respectively.
TL;DR: It is shown that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)‐mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in human kidney‐transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal cal bindin‐D 28 kDa protein level was found in most of the kidney biopsy sections.
Abstract: Using two-dimensional gel electrophoresis (2-DE), we recently discovered an association between decreased calcium-binding protein, calbindin-D 28 kDa, urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney. This observation prompted us to investigate kidney tissues of other species, including man. In this paper we show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)-mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in CsA-treated human kidney-transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin-D 28 kDa protein level was found in most of the kidney biopsy sections. The present results strongly suggest that calbindin is a marker for CsA-nephrotoxicity. The discovery of calbindin-D 28 kDa being involved in CsA toxicity has evolved from the application of 2-DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology. Considering the current improvements in proteome methods it is expected that high throughput proteomics will become an indispensable tool in preclinical safety testing.
TL;DR: Recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins are summarized and the emerging field of microfluidics as a front end to mass spectrometry (MS).
Abstract: Analytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever-increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method. In this review article we summarize recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins. Specifically, we review recent advances in the development and application of capillary electrophoresis-mass spectrometry (CE-MS) and on-line analyte concentration techniques, and introduce the emerging field of microfluidics as a front end to mass spectrometry (MS).
TL;DR: The study has demonstrated significant alterations in quantitative protein expression in the DCM heart which would have serious implications for myocyte function and might be explained by altered protease activity in DCM which could exacerbate contractile dysfunction in the failing heart.
Abstract: The aim of the investigation was to determine whether there are specific global quantitative and qualitative changes in protein expression in heart tissue from patients with dilated cardiomyopathy (DCM) compared with ischaemic heart disease and undiseased tissue. Two-dimensional (2-D) polyacrylamide gel electrophoresis and computer analysis was used to study protein alteration in DCM biopsy material (n=28) compared with donor heart biopsy samples (n=9) and explanted hearts from individuals suffering from ischaemic heart disease (IHD; n = 21). A total of 88 proteins displayed decreased abundance in DCM versus IHD material while five proteins had elevated levels in the DCM group (p<0.01). The most prominent changes occurred in the contractile protein myosin light chain 2 and in a group of proteins identified as desmin. These changes do not appear to be artefactual degradation events occurring during sample processing. These proteins are not apparent in electrophoretic separations of vascular tissue or cultured endothelial cells, mesothelial cells or cardiac fibroblasts, which are clearly distinguishable from the 2-D protein patterns of whole heart and of isolated cardiac myocytes and do not appear to reflect variations in the cellular composition of biopsy samples. The different protein patterns observed in cardiomyopathy showed no obvious relationship with New York Heart Association (NYHA) functional class or haemodynamic parameters. The study has demonstrated significant alterations in quantitative protein expression in the DCM heart which would have serious implications for myocyte function. These changes might be explained by altered protease activity in DCM which could exacerbate contractile dysfunction in the failing heart.
TL;DR: Enantiomeric separations of D‐ and L‐dansyl phenylalanines were achieved in both OT‐LC and OT‐CEC modes with good selectivity and efficiencies and effects of polymerization conditions on column preparation and chromatographic performance were studied.
Abstract: Chiral separations employing molecular imprint polymer (MIP) stationary phases in both open tubular liquid chromatography (OT-LC) and capillary electrochromatography (OT-CEC) are demonstrated. MIPs are highly crosslinked polymers containing spatial and functionality memory of template molecules which provide a higher degree of selectivity when used as stationary phases for chromatographic separations. Thin films of molecular imprinted polymers bonded to the inner walls of 25 microm ID fused-silica capillaries were prepared using an in situ polymerization technique developed in our laboratory that allows the use of conventional fused-silica capillaries with polyimide outer coatings. The success rate in preparing such open tubular columns was about 70%. Methacrylic acid and 2-vinyl pyridine were chosen as functional monomers, and either ethylene dimethacrylate or trimethylol propane trimethacrylate was used as the crosslinker. Toluene was employed as the porogen. Effects of polymerization conditions on column preparation and chromatographic performance were studied. Enantiomeric separations of D- and L-dansyl phenylalanines were achieved in both OT-LC and OT-CEC modes with good selectivity and efficiencies. Both types of separations may be performed on the same column using a single commercial instrument.
TL;DR: This study shows how the use of exon‐primed, intron‐crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level.
Abstract: In this study we show how the use of exon-primed, intron-crossing (EPIC) polymerase chain reaction (PCR) of a diploid intronic region, in conjunction with temperature gradient gel electrophoresis (TGGE), can be used to detect and rapidly assess allelic variation at the nucleotide level. We developed passerine-specific primers to amplify and sequence a 762 bp region including the second intron of the myoglobin gene in the Gouldian Finch, Erythrura gouldiae. A POLAND plot based on this sequence indicated that TGGE in combination with heteroduplex analysis (TGGE/HA) should reveal nucleotide variation in the 160 bp low-melting domain. Sequencing of the entire fragment from 19 Er. gouldiae revealed five nucleotide substitution differences within the low-melt domain, all of which could be detected and differentiated by TGGE/HA, and an additional substitution in a section of the high-melt domain which characterised another allele. A total of 181 individuals from four populations were screened for these six alleles.