Proteomic basis for the possible use of lymphocytes for metabolic screenings

Summary. The advent of proteomics has provided a tool for the concomitant identification and determination of a large series of proteins using two-dimensional gel electrophoresis with subsequent mass spectrometrical analysis. We tried an approach to analyse the high abundance enzyme proteome of a lymphocytic cell line.Immortalised lymphocytes were grown in RPMI 1640 in the presence of glutamine, harvested and the 100,000×g supernatant of the homogenate was applied on two-dimensional gel electrophoresis with subsequent in-gel digestion of protein spots and MALDI-TOF (Matrix-associated laser desorption/ionization mass spectroscopy) analysis of resulting peptides using specific software.A series of 57 metabolic enzymes were identified including enzymes of carbohydrate, amino acid, purine and intermediary metabolism.We are presenting a tool for the analysis of metabolic systems including enzyme deficiencies at the protein level with the advantage of unambiguous identification of proteins and thus complementing enzyme activity determinations.J. E. Oh and K. Krapfenbauer have been equally contributing to the work.     

Characteristics of basal taurine release in the rat striatum measured by microdialysis

Summary. Taurine is a sulfur-containing amino acid thought to be an osmoregulator, neurotransmitter or neuromodulator in the brain. Our objective was to establish how much taurine is released in the striatum and examine the mechanisms controlling extracellular taurine concentrations under resting conditions. The experiments were made on rats by microdialysis in vivo. Changes in taurine were compared with those in glutamate, glycine and the non-neuroactive amino acid threonine. Using the zero net flux approach we showed the extracellular concentration of taurine to be 25.2±5.1M. Glutamate was increased by tetrodotoxin and decreased by Ca²⁺ omission, glycine and threonine were not affected and both treatments increased extracellular taurine. The basal taurine release was increased by the taurine transport inhibitor guanidinoethanesulfonate and reduced by the anion channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid.     

Threonine metabolism in Japanese quail liver

Summary. In general, threonine is metabolized by reaction catalyzed by threonine-3-dehydrogenase (TDH), threonine dehydratase (TH) or threonine aldolase (TA). The activities of these three enzymes were compared in the liver of Japanese quails and rats. The animals were fed a standard or threonine rich-diet, or fasted for 3 days. The specific activity of TDH in the liver from quail fed a standard diet was 11 times higher than that in the liver from rats fed a standard diet. The TDH activities in the livers of the fasting and 5% threonine-rich diet groups of quail were 3 and 2 times higher than those in the livers from quail fed the standard diet, respectively. The TH activity in the liver of rats fed a standard diet was 14 times higher than that in the liver of quail fed a standard diet. The TH activity in the rat liver after fasting was 2.3 times higher than that of the standard diet control. The activity of TA in the livers of rat and quail were so low that its role in threonine metabolism in both animals seemed to be negligible. These results suggest that threonine is a ketogenic amino acid in the quail liver, while it is a glucogenic in the rat liver.     

Strategies for the enrichment and identification of basic proteins in proteome projects

Two-dimensional gel electrophoresis (2-DE) is currently the method of choice for separating complex mixtures of proteins for visual comparison in proteome analysis. This technology, however, is biased against certain classes of proteins including low abundance and hydrophobic proteins. Proteins with extremely alkaline isoelectric points (pI) are often very poorly represented using 2-DE technology, even when complex mixtures are separated using commercially available pH 6-11 or pH 7-10 immobilized pH gradients. The genome of the human gut pathogen, Helicobacter pylori, is dominated by genes encoding basic proteins, and is therefore a useful model for examining methodology suitable for separating such proteins. H. pylori proteins were separated on pH 6-11 and novel pH 9-12 immobilized pH gradients and 65 protein spots were subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry, leading to the identification of 49 unique proteins. No proteins were characterized with a theoretical pI of greater than 10.23. A second approach to examine extremely alkaline proteins (pI > 9.0) utilized a prefractionation isoelectric focusing. Proteins were separated into two fractions using Gradiflow technology, and the extremely basic fraction subjected to both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography (LC) - tandem mass spectrometry post-tryptic digest, allowing the identification of 17 and 13 proteins, respectively. Gradiflow separations were highly specific for proteins with pI > 9.0, however, a single LC separation only allowed the identification of peptides from highly abundant proteins. These methods and those encompassing multiple LC 'dimensions' may be a useful complement to 2-DE for 'near-to-total' proteome coverage in the alkaline pH range.     

Application of displacement chromatography for the analysis of a lipid raft proteome

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC–MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC–MS approach.     

Depletion of the high-abundance plasma proteins

Summary. Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.Current address: Foundation for Biomedical Research of the Academy of Athens, Greece     

Effect of pH and salts on the binding of free amino acids to the corn protein zein studied by thin-layer chromatography

Summary. The interaction of free amino acids with the corn protein zein was studied by thin-layer chromatography carried out on cellulose layers covered with zein and the effect of pH and salts on the strength of interaction was elucidated. Only the binding of Arg, His, Lys, Orn and Trp to zein was verified, other amino acids were not retained. Retention of Arg, His, Lys and Orn decreased linearly with increasing concentration of salts the mobile phase indicating the hydrophilic character of amino acid–zein interaction. Both alkaline and acidic pH influenced the strength of binding. Principal component analysis indicated the different character of the influence of pH and salts on the interaction. The results suggest that these amino acid residues may account for the binding of other peptides and proteins to zein.     

Flow field-flow fractionation: a pre-analytical method for proteomics

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.     

Proteomic profiling reveals compartment-specific, novel functions of ascidian sperm proteins

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X-100-soluble and -insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS-PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non-redundant proteins were identified by mass spectrometry (MALDI-TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm-specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca(2+)-binding proteins in axonemes, localization of testis-specific serine/threonine kinase, and the presence of G-protein coupled signaling and ubiquitin pathway in sperm flagella.     

Improvement of the two-dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.     

Proteins interacting with the tuberous sclerosis gene products

Summary. Tuberous sclerosis (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 to 10000 individuals. The genes, TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for TSC. Since their identification 1997 and 1993 respectively, a variety of different functions have been described for the TSC gene products. Hamartin and tuberin form a complex, providing a tentative explanation for the similar disease phenotype in TSC patients with mutations in either of these genes. In addition, associations of hamartin or tuberin with several different proteins have been demonstrated. In this review, we summarize the current knowledge on hamartin- and tuberin-interacting proteins and discuss their role for the understanding of the functions of the TSC gene products.     

Inhibition of the L-dopa transport system in human epidermal Langerhans cells by omeprazole and its analogues

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) transport into human Langerhans cells (LC) occurs by a saturable mediation. This plasma membrane agency is, due to its characteristics, distinguishable from systems transporting other neutral, cationic and anionic amino acids into other cells and serves to catalyze the flow of L-dopa, only, into LC. The uphill operation of this L-dopa transport system is believed to occur by down-gradient countermigration of H⁺. Due to the uniqueness of the L-dopa transport system, the widely used analogue inhibition approach was not applicable. Instead we studied omeprazole and its analogues in our search for suitable inhibitory candidates. Omeprazole and most of its analogues were indeed inhibitory in the concentration range 1–100mol/L. Conspicuously, the compounds with strongest polarity were least inhibitory. The inhibitory pattern displayed by omeprazole and the other analogues on L-dopa uptake in LC corresponded to some extent to what has been observed previously for purified H⁺,K⁺-ATPase from tubulovesicles of the stomach. No effects of the inhibitors were registered on energy charge and lactate production of epidermal biopsies, nor were any gross alterations of ultrastructure of LC noticed.     

Resonance light‐scattering enhancement effect of the protein–Y3+–TTA–SLS system and its analytical application

In this paper, a sensitive resonance light scattering (RLS) method for the determination of protein is reported. In the Tris–HCl (pH 7.50) buffer, protein enhanced the RLS intensity of the Y³⁺–2‐thenoyltrifluoroacetone (TTA)–sodium dodecyl sulphate (SLS) system. The enhanced RLS intensities were in proportion to the concentrations of proteins in the range 8.0 × 10^?9–1.0 × 10^?5 g/mL for BSA, 1.0 × 10^–8–1.0 × 10^?5 g/mL for HSA and 1.0 × 10^–8–1.0 × 10^?6 g/mL for EA, and their detection limits were 5.0, 5.4 and 6.7 ng/mL, respectively. Actual samples were satisfactorily determined. The interaction mechanism was also studied. Copyright © 2008 John Wiley & Sons, Ltd.     

Peroxynitrite-dependent modifications of tyrosine residues in hemoglobin. Formation of tyrosyl radical(s) and 3-nitrotyrosine

Summary. Although peroxynitrite is believed to be one of the most efficient tyrosine-nitrating species of biological relevance so far identified, its nitration efficiency is nevertheless limited. In fact, the nitrating species formed through peroxynitrite decay are caged radicals (OH/NO₂ or, in the presence of carbon dioxide, CO₃ ^–/NO₂) and the fraction that escapes from the solvent cage does not exceed 30–35%. One exception may be represented by metal-containing compounds that can enhance the formation of nitrotyrosine through a bimolecular reaction with peroxynitrite. Moreover, if the metal is also regenerated in the reaction, the compound is considered a nitration catalysts and the yield of tyrosine nitration enhanced several fold. Examples of peroxynitrite-dependent nitration catalysts are the Mn-superoxide dismutase, some cytochromes and several metalloporphyrins. On the contrary, it has been claimed that some hemoproteins are scavengers of peroxynitrite and play a role in limiting its biodamaging and bioregulatory activity. In this review, we discuss the case of hemoglobin, which is probably the major target of peroxynitrite in blood. This protein has been reported to protect intracellular and extracellular targets from peroxynitrite-mediated tyrosine nitration. This property is shared with myoglobin and cytochrome c. The possible mechanisms conferring to these proteins a peroxynitrite scavenging role are discussed.Present address: Laboratorio di Tossicologia Applicata ed Ecotossicologia, Istituto Superiore di Sanità, Rome, Italy.     

Morphinome – proteome of the nervous system after morphine treatment

Summary. Proteome is a natural consequence of the post-genome era when the HUGO project (Human Genome Organization) has almost been completed. Here, a specifically aimed proteome in drug dependence – morphinome, is described, including tasks, strategies and pitfalls of the methodology.     

Homology modeling reveals the structural background of the striking difference in thermal stability between two related [NiFe]hydrogenases

Hydrogenases are redox metalloenzymes in bacteria that catalyze the uptake or production of molecular hydrogen. Two homologous nickel–iron hydrogenases, HupSL and HydSL from the photosynthetic purple sulfur bacterium Thiocapsa roseopersicina, differ substantially in their thermal stabilities despite the high sequence similarity between them. The optimum temperature of HydSL activity is estimated to be at least 50 °C higher than that of HupSL. In this work, homology models of both proteins were constructed and analyzed for a number of structural properties. The comparison of the models reveals that the higher stability of HydSL can be attributed to increased inter-subunit electrostatic interactions: the homology models reliably predict that HydSL contains at least five more inter-subunit ion pairs than HupSL. The subunit interface of HydSL is more polar than that of HupSL, and it contains a few extra inter-subunit hydrogen bonds. A more optimized cavity system and amino acid replacements resulting in increased conformational rigidity may also contribute to the higher stability of HydSL. The results are in accord with the general observation that with increasing temperature, the role of electrostatic interactions in protein stability increases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00894-001-0071-8.Electronic Supplementary Material available.     

Expression of proteasomal proteins in ten different tumor cell lines

Summary. Controlled intracellular protein degradation is crucial for the maintenance of normal cell functions. An evolving concept claims that alterations in the exact timely degradation of proteins involved in growth control, apoptosis, signaling and differentiation contribute to carcinogenesis. This tightly regulated process is facilitated by the ubiquitin-26S proteasome system, a multi-enzyme complex, and inhibitors of this pathway have already been developed as potential anticancer agents.In order to generate proteasomal protein expression patterns of tumor cells and to provide an analytical tool we applied two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MALDI-TOF-TOF with LIFT technology) in ten individual tumor cell lines (Saos-2; SK-N-SH; HCT-116; Caov3; A-549; HL60; A-673; A-375; MCF-7; HeLa) widely used in tumor research. A series of 39 proteasomal/proteolytic proteins was unambiguously identified by this proteomic approach, comprising proteins of the 20S core complex, the 19S regulatory complex, the 11S regulator, components of the ubiquitin pathway and proteases.Construction of individual protein maps by 2-DE and mass spectrometry provides an analytical tool and reference base for studying the pivotal importance of the proteasome and other proteolytic enzymes in tumor cells, independent of antibody availability and specificity. This preliminary database enables for designing studies in this area of research and reveals proteins that can be used as targets for new therapeutic strategies.     

Polyamines biosynthesis and oxidation in free-living amoebae

Summary. In this paper we describe the polyamine biosynthesis and oxidation processes, giving an overview about recent results in free-living Amoebae.The protozoa polyamine levels are different in comparison with mammalian cells. Also, the polyamine levels in protozoa cells change if these species are pathological or not for the human beings. All the amoeba strains show high concentrations of 1,3-diaminopropane (DAP), spermidine and acetylspermidine while spermine is absent. In these amoeba a considerable polyamine oxidase activity has been found, which acts on N⁸-acetylspermidine, but not on free polyamines. This enzyme is responsible, together with polyamine acetylase, of DAP synthesis whose function is not well known.