High resolution mini-two-dimensional gel electrophoresis of yeast ribosomal proteins

By applying two-dimensional gel electrophoresis (basic urea/acidic urea) in a mini-form yeast ribosomal proteins were separated with a high resolution. On basis of this separation pattern a standard nomenclature for the yeast ribosomal proteins is proposed.     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Affinity capillary electrophoresis for the screening of novel antimicrobial targets

The increasing number of multiantibiotic-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), requires the development of novel chemotherapies that are structurally distinct and exempt from current resistance mechanisms. Bioinformatics data mining of microbial genomes has revealed numerous previously unexploited essential open reading frames (ORFs) of unknown biochemical function. The potential of these proteins as screening targets is not readily apparent because most screening technologies rely on knowledge of biological function. To address this problem, the authors employed affinity capillary electrophoresis (ACE) to identify antimicrobial compounds that bound the novel target YihA. Screening a small-molecule library of 44,000 compounds initially identified 115 binders, of which 76% were confirmed. Furthermore, the ACE assay distinguished diverse compounds that possessed drug-like properties and antimicrobial activity against drug-resistant clinical isolates. These data validate ACE as a valuable tool for the fast, efficient detection of specific binding molecules that possess biological activity.     

Affinity labelling of yeast ribosomal peptidyl transferase

Summary Using p-nitrophenylcarbamyl-phenylananyl-tRNA (PNPC-Phe-tRNA) and N-Iodoacetyl-phenylalanyl-tRNA as affinity labels we have attempted to identify the components of the aminoacyl-tRNA binding sites located in the vicinity of the peptidyl transferase centre of the yeast ribosome. Both Phe-tRNA derivatives bind to the ribosomal A-site in the presence of 20 mM Mg⁺⁺ ion concentration and can be translocated to the ribosomal P-site in the presence of elongation factor. After the labels have been allowed to react covalently with ribosomes they were found associated with the large ribosomal subunit. Proteins L36, L43, L42, L29, L2, L17/18, L19/20 and proteins L26, L38, L22/23, L7/9, L4/6, L36, L11, L43, L39 were labelled in samples treated with PNPC-Phe-tRNA and N-Iodoacetyl-Phe-tRNA respectively. In contrast, when only the components of the ribosomal P-site were analysed by reacting the treated particles with puromycin fewer spots were labelled, corresponding to proteins L36 and L19/20 using PNPC-Phe-tRNA and proteins L4/6, L36, and L43 using N-Iodoacetyl-Phe-tRNA.     

Studies of ribosomal proteins of yeast species and their hybrids gel electrophoresis and immunochemical cross-reactions

Summary The cytoplasmic ribosomal proteins (r-proteins) of seventeen yeast species of the genera Saccharomyces and Kluyveromyces were analyzed by one-dimensional gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The electrophoretic patterns of cytoplasmic r-proteins from different species display extensive differences in both the 40S and the 60S subunit. Relatedness of species suggested by r-protein patterns correlates well with that based on DNA/DNA homology (Bicknell and Douglas 1970). Immunochemical cross-reactions and antibiotic susceptibility tests were also used to compare different species.Analyses of r-proteins from two different interspecific hybrids showed that their ribosomes were hybrid, containing r-proteins from both parents. These findings are discussed in relation to the evolution of yeast species and the regulation of expression of r-proteins in cucaryotes.     

Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast

Summary Two mitochondrial ribosomal proteins of yeast (Saccharomyces cerevisiae) were purified and their N-terminal amino acid sequences determined. The sequence data were used for the synthesis of oligonucleotide probes to clone the corresponding genes. Thus, the genes for two proteins, termed YMR-31 and YMR-44, were cloned and their nucleotide sequences determined. From the nucleotide sequence data, the coding region of the gene for protein YMR-31 was found to be composed of 369 nucleotide pairs. Comparison of the amino acid sequence of protein YMR-31 and the one deduced from the nucleotide sequence of its gene suggests that it contains an octapeptide leader sequence. The calculated molecular weight of protein YMR-31 without the leader sequence is 12792 dalton. The gene for protein YMR-44 was found to contain a 147 bp intron which contains two sequences conserved among yeast introns. The length of the two exons flanking the intron totals 294 nucleotide pairs which can encode a protein with a calculated molecular weight of 11476 dalton. The gene for protein YMR-31 is located on chromosome VI, while the gene for protein YMR-44 is located on either chromosome XIII or XVI.     

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.     

A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

Ribosomal proteins from the yeast Saccharomyces cerevisiae were separated, on a preparative scale, by ion-exchange h.p.l.c. Proteins from the small and large ribosomal subunits were resolved, respectively, into 33 and 23 peaks, and most of the proteins present in these peaks were identified by using one- and two-dimensional gel electrophoresis. Several of the peaks appeared to contain a single protein uncontaminated by other species. Ribosomal proteins were also separated by using reverse-phase h.p.l.c. Analysis of the peaks resolved indicated that the order of elution for the proteins of both ribosomal subunits is, in certain cases, different for each of the two h.p.l.c. techniques used. Thus a combination of the two chromatographic methods employed here has the potential to facilitate the rapid and preparative separation of each of the proteins present in yeast ribosomes.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

Physico-chemical characterization of proteins by capillary electrophoresis

The electrophoretic mobility of proteins was successfully determined by means of capillary electrophoresis (CE) with various background electrolytes (BGEs). The objective was focused on the variation in BGE physico-chemical composition and the consequential impact on the observed protein charge. Experimental and calculated mobilities, according to Henry's equation, versus ionic strength have been compared. For positively-charged lysozyme, a good agreement between observed and calculated mobilities was observed using triethanolamine chloride at pH 7.0 as the BGE. Mobility close to zero was shown using borate (pH 8.0) and phosphate (pH 7.0) at a low ionic strength of about 20 mmol l⁻¹, and as a consequence, specific adsorption of oxyanions was evidenced. Lysozyme retention in the case of reversed-phase high-performance liquid chromatography (RP-HPLC) was decreased by the presence of phosphate ions. CE and HPLC are complementary tools for characterizing the behaviour of lysozyme. On the other hand, the mobility of the negatively-charged α-lactalbumin remained constant as regards phosphate at pH 7.0 in the 20–200 mmol l⁻¹ range, contrary to the decrease that had been expected with the increasing ionic strength. β-Lactoglobulin exhibited increasingly lower mobilities than those expected of boric acid/borate at pH 7.0 and 8.0 (I=20 mmol l⁻¹).     

Optimization of capillary coating by hydroxyethyl methacrylate for capillary zone electrophoresis of proteins

This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water-methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).     

Affinity electrophoresis of proteins interacting with Blue dextran.

Interaction of several enzymes (pyruvate kinase, myokinase, creatine kinase, aldolase, malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase) and other proteins (bovine serum albumin and ovalbumin) with Blue Dextran was studied by means of affinity electrophoresis in polyacrylamide gels. A decrease of electrophoretic mobility of enzymes in affinity gels was dependent on Blue Dextran concentration and in some cases, dissociation constants of the protein-immobilized dye complexes could be calculated. Affinity electrophoresis in the presence of Blue Dextran reveals in some cases additional bands of isoenzymes, as compared with the control gels (without Blue Dextran).     

Micro-scale two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins

Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.     

Synthesis of ribosomal proteins during the yeast cell cycle

The synthesis of ribosomal proteins during the cell division cycle of Saccharomyces cerevisiae has been examined. A technique was utilized whereby cells in unique phases of the cell cycle were selected from an asynchronous culture after the period of pulse labeling. Some of the proteins of the small and large ribosomal subunits were synthesized continuously throughout the cell cycle and there was no evidence of discontinuous synthesis for any of the ribosomal proteins.     

Size-exclusion liquid chromatography and capillary electrophoresis of pollen allergens

Allergens from the pollen of Phleum pratense, Dactylis glomerata. Arrhenatherum elatius, Secale cereale, Lolium perrene and Festuca sp. were analysed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). SEC was used for the determination of the molecular masses of main allergens. A CE method, using either 150 mmol/1 phosphoric acid (pH 1.8) or a micellar system consisting of 50 mmol/l sodium dodecyl sulphate-20 mmol/l borate (pH 9.35), was developed as a rapid and efficient alternative to SEC, especially for process control of allergenic preparations. The results obtained by the two methods confirmed similarities in the structures of the studied pollen allergens.     

Affinity chromatography of bull seminal proteins on mannan-Sepharose

4-Hydroxynon-2-enal (4-HNE) is one of the major aldehydic products of lipid peroxidation (LPO) and is involved in a number of pathophysiological processes. Since LPO products are useful indicators for oxidative stress in vivo, a number of detection methods for LPO products in biological tissues were developed. However, none of these methods is presently used in clinical settings. In order to introduce LPO products as biomarkers in clinical studies a suitable GC-MS method for 4-HNE detection was adapted to meet clinical requirements. As one result, the minimal sample volume could be decreased to 50 microl of plasma so that the method might even be suitable for pediatric purposes. The best internal standard (I.S.) for 4-HNE detection by GC-MS 9,9,9-D(3)-4-hydroxynon-2-enal was introduced by van Kuijk et al. [Anal. Biochem., 224 (1995) 420]. However, because of its limited availability, benzaldehyde-ring-d(5), 4-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde were tested to find an alternative. Out of these three, 4-hydroxybenzaldehyde was shown to serve best as I.S. To examine the applicability of the adapted method, tests on the stability of 4-HNE in samples during storage were carried out. It was shown that plasma samples need to be stored at -80 degrees C or less to avoid greater loss of 4-HNE. Samples with 4-HNE concentrations close to the physiological level were shown to be stable over 22 months at -80 degrees C. The introduction of a new and easily available I.S., reduction of the sample volume, and information about sample stability provided by this study facilitate 4-HNE determination in most clinical settings.     

Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing

Strategies reported for the separation of proteins in capillary zone electrophoresis and capillary isoelectric focusing are reviewed. The strategies are grouped into two categories: coated capillaries and buffer/sample additives. Success attained with each case and also, more importantly, the limitations of the methodology are discussed. Recent results from our own laboratory in the area of capillary isoelectric focusing in uncoated, fused silica capillaries using additives are summarized. The advantages and disadvantages of coated columns vs. additives are delineated.