Biomedical subjects

Pier Giorgio Righetti

Publications and source records attributed to Pier Giorgio Righetti.

At least 19 recordsLinked to original sources

PubMed2005

Prefractionation techniques in proteome analysis: the mining tools of the third millennium.

The present review deals with prefractionation protocols used in proteomic investigation in preparation for mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) map analysis. Briefly, reported methods focus on cell organelle differential centrifugation and on chromatographic approaches, to continue in extenso with a panoply of electrophoretic methods. In the case of chromatography, procedures useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins, revealed several hundreds of new species, previously undetected in unfractionated samples. Novel chromatographic prefractionation methods are also discussed such as a multistaged fractionation column, consisting in a set of immobilized chemistries, serially connected in a stack format (an assembly of seven blocks), each capable of harvesting a given protein population. Such a method significantly simplifies the complexity of treated samples while concentrating species, all resulting in a larger number of visible proteins by MS or 2-DE. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separation are multichamber apparatus, such as the multicompartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads. Multicup device electrophoresis and several others, exploiting the conventional technique of carrier ampholyte focusing, are reviewed. This review also reports approaches for sample treatments in order to detect low-abundance species. Among others, a special emphasis is made on the reduction of concentration difference between proteins constituting a sample. This latter consists in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of 20 amino acids, resulting in millions of different structures. When these beads are impregnated with complex proteomes (e.g., human sera) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility to evidence low-abundance species. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Cell Fractionation↗

PubMed2005

Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administration.

In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36 spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine.

Animals↗

PubMed2005

Two-dimensional mapping as a tool for classification of green coffee bean species.

Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two-dimensional (2-D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and beta-mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 microL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3-(3-cholamidopropyldimethyl-amino)-1-propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2-D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up-regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down-regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix-assisted laser desorption/ionisation-time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase-high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed.

Chromatography, High Pressure Liquid↗

PubMed2005

Numerical approaches for quantitative analysis of two-dimensional maps: a review of commercial software and home-made systems.

The present review attempts to cover a number of methods that have appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this review to conventional two-dimensional (2-D) map analysis which couples orthogonally a charge-based step (isoelectric focusing) to a size-based separation step (sodium dodecyl sulfate-electrophoresis). The first and oldest method applied to 2-D map data reduction is based on statistical analysis performed on sets of gels via powerful software packages, such as Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately running a number of replicas for control and treated samples. The two sets of data are then merged and compared via a number of software packages which we describe. In addition to commercially-available systems, a number of home made approaches for 2-D map comparison have been recently described and are also reviewed. They are based on fuzzyfication of the digitized 2-D gel image coupled to linear discriminant analysis, three-way principal component analysis or a combination of principal component analysis and soft-independent modeling of class analogy. These statistical tools appear to perform well in differential proteomic studies.

Cluster Analysis↗

PubMed2005

Isoelectric beads for proteome pre-fractionation. II: experimental evaluation in a multicompartment electrolyzer.

Proteome pre-fractionation in multicompartment electrolyzers is proposed here, with substantial modifications as compared to the standard technique. First of all, the classical isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical continuous membrane barriers). Secondly, the isoelectric beads are not made in the conventional manner, with ionic acrylamide derivative monomers throughout their central core, but are composed of a hard, ceramic core, coated with an amphoteric buffering polymer. This minimizes mass transfer resistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reduced separation times, of the order of ca. 3 h, are required for developing steady-state patterns, as compared to the lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operating with isoelectric membranes. Examples of separation of standard marker proteins, as well as entire Escherichia coli lysates and human serum proteins, are given. The obtained fractions are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by surface enhanced laser desorption/ionization mass spectrometry.

Blood Proteins↗

PubMed2005

Amphoteric, buffering chromatographic beads for proteome prefractionation. I: theoretical model.

The possibility is reported here of fractionating proteins on amphoteric, buffering resins via ion-exchange chromatography. A given protein's adsorption to a particular amphoteric buffering resin is characterized by a bell-shaped curve in which the maximum protein binding capacity is observed at an optimum pH value lying approximately midway between the isoelectric point values (pI) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the pI of either the protein or exchanger. For instance, on beads of pI equal to 8, four proteins, two acidic (bovine albumin and ovalbumin) and two basic (cytochrome c and lysozyme), exhibit binding curves reaching zero values for the whole set when the exchanger is conditioned at pH 8.0. Away from the pI, and on both sides of the pH scale, the bell-shaped adsorption curves reach a maximum, for each protein, at a pH located at the midpoint between the pI values of each protein and that of the exchanger, and decline steadily to reach zero at the pI value of each protein species. Separation of model proteins using different amphoteric buffering resins of various pI was possible at different pH values according to both the pI of the proteins and of the exchangers. It was also demonstrated, using surface enhanced laser desorption/ionization mass spectrometry and two dimensional electrophoretic mapping, that separation of an Escherichia coli cell lysate on columns packed with amphoteric buffering resins of different pI and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a pI below the buffer pH (the 'adsorbed' fraction), and on the other, of alkaline proteins possessing a pI above the pH of the buffer (the 'unadsorbed' fraction). This approach represents an attractive addition and/or alternative to the armory of protein pre-fractionation techniques currently employed in proteomics.

Albumins↗

PubMed2005

Differential accumulation of Lhcb gene products in thylakoid membranes of Zea mays plants grown under contrasting light and temperature conditions.

In higher plants many different genes encode Lhcb proteins that belong to a highly conserved protein family. Evolutionary conservation of this genetic redundancy suggests that individual gene products play different roles in light harvesting and photoprotection depending on environmental conditions. We have tested the hypothesis that expression/accumulation of individual light harvesting complex (Lhc) proteins depends on plant growth conditions. Zea mays plants were grown in different temperature (13 degrees C vs. 24 degrees C) and light (high vs. low) conditions. The thylakoid membranes were isolated and fractionated by sucrose gradient and the protein content of the different bands was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences were found in the accumulation of both the major light harvesting complex of photosystem II (LHCII) complexes and the minor antenna chlorophyll proteins CP29, CP26 and CP24. In particular, temperature seems to play a major role in driving the expression/accumulation of the different proteins: the LHCII/minor antenna ratio increases with decreasing temperature. The pigment composition and the spectroscopic properties of LHCII complexes isolated from low temperature grown plants are significantly different from those of LHCII purified from high temperature grown plants. Two-dimensional maps show that different LHCII proteins are accumulated at different levels depending on growth conditions. Moreover the low temperature/high light grown plants show an increased value of nonphotochemical quenching. These results suggest a specific role of different LHCII complexes in the organization of the potosystem II and photoprotection.

Chemical Fractionation↗

PubMed2004

Crystal structure of chicken liver basic fatty acid-binding protein complexed with cholic acid.

Two paralogous groups of liver fatty acid-binding proteins (FABPs) have been described: the mammalian type liver FABPs and the basic type (Lb-FABPs) characterized in several vertebrates but not in mammals. The two groups have similar sequences and share a highly conserved three-dimensional structure, but their specificity and stoichiometry of binding are different. The crystal structure of chicken Lb-FABP complexed with cholic acid and that of the apoprotein refined to 2.0 A resolution are presented in this paper. The two forms of the protein crystallize in different space groups, and significant changes are observed between the two conformations. The holoprotein binds two molecules of cholate in the interior cavity, and the contacts observed between the two ligands can help to explain the reason for this stoichiometry of binding. Most of the amino acids involved in ligand binding are conserved in other members of the Lb-FABP family. Since the amino acid sequence of the Lb-FABPs is more similar to that of the bile acid-binding proteins than to that of the L-FABPs, the possibility that the Lb-FABPs might be more appropriately called liver bile acid-binding proteins (L-BABPs) is suggested.

Amino Acid Sequence↗

PubMed2004

Surfing silica surfaces superciliously.

The present mini-review summarizes the experience gathered by our group in developing different classes of novel quaternarized heterocyclic compounds able to modulate and reverse the electroendoosmotic flow (EOF) in a most peculiar manner. The first class comprises mono-salt compounds, with the determinant omega-iodoalkyl chains of different lengths (typically C4-C8), able to be adsorbed by silicas, at alkaline pH, and spontaneously alkylate ionised silanols, thus becoming covalently affixed to it. The second class is constituted by di-salt compounds, attached at the termini of an alkyl chain of variable lengths (here too, typically, C4-C8). This second class is unable to bind covalently silica surfaces, although, in thin-layer chromatography, it exhibits an extraordinary affinity for silica beads, contrary to the first one. On the basis of the strikingly different behaviour, structural rules are derived for the minimum requirements for general classes of amines to bind to silica walls and modify EOF. For compounds unable to bind covalently to the wall, the most important structural motif is two quaternary nitrogens spaced apart by a C4 chain: this seems to be the average distance (i.e., 0.8 nm) between two adjacent, ionized silanols for a snug fit. The other structural binding motif is the "hydrophobic decoration", i.e., the ratio of charged groups to alkyl residue in the various amines; amines with high levels of such alkane groups (i.e., with higher hydrophobicity), seem to bind more tenaciously to the wall, probably due to hydrophobic interaction not to the wall but among the amine derivatives themselves, when carpeting the silica.

Silicon Dioxide↗

PubMed2004

Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches.

The present review attempts to cover a number of methods that appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this excursus only to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (sodium dodecyl sulfate (SDS)-electrophoresis). The first and oldest method applied in 2D mapping is based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately-running a number of replicas for control and treated samples, the merging and comparing between these two sets of data being accomplished via the softwares just mentioned. Recent developments permit analyses on a single gel containing mixed samples differentially labelled and resolved by either fluorescence or isotopic means. In one approach, a set of fluorophors, called Cy3 and Cy5, are selected for differentially tagging Lys residues, via a "minimal labelling" protocol. A variant of this, adopts a newer set of fluorophors, also of the Cy3 and Cy5 type, reacting on Cys residues, via a strategy of "saturation labelling". There are at present two methods for quantitative proteomics in a 2D gel format exploiting stable isotopes: one utilizes tagging Cys residues with [2H0]/[2H3]-acrylamide; the other one, also based on a Cys reactive compound, exploits [2H0]/[2H4] 2-vinylpyridine. The latter reagent achieves 100% efficiency coupled to 100% specificity. The advantages and limitations of the various protocols are discussed.

Amino Acid Sequence↗

PubMed2004

Proteomic analysis of an orthotopic neuroblastoma xenograft animal model.

Neuroblastoma is the most common extracranial solid tumour of childhood and comprises up to 50% of malignancies among infants. There is a great need of designing novel therapeutic strategies and proteome analysis is one approach for defining markers useful for tumour diagnosis, as well as molecular targets for novel experimental therapies. We started by comparing healthy adrenal glands (which are the election organs developing primary neuroblastoma, NB, tumours) and adrenal glands carrying primary NB tumours, taken from nude mice. Standard maps of healthy and tumour samples were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups, were analysed by MALDI-TOF mass spectrometry and 14 of these spots were identified so far. Among these proteins, of particular interest are the down-regulated proteins adrenodoxin (21-folds), carbonic anhydrase III (eight-folds) and aldose reductase related protein I (eight-folds), as well as the up-regulated protein peptidyl-propyl cis-trans isomerase A (five-folds). Moreover new proteins, which were absent in control samples, were expressed in tumour samples, such as nucleophosmin (NPM) and stathmin (oncoprotein 18).

Animals↗

PubMed2004

Identification of the regulatory proteins in human pancreatic cancers treated with Trichostatin A by 2D-PAGE maps and multivariate statistical analysis.

In this paper, principal component analysis (PCA) is applied to a spot quantity dataset comprising 435 spots detected in 18 samples belonging to two different cell lines (Paca44 and T3M4) of control (untreated) and drug-treated pancreatic ductal carcinoma cells. The aim of the study was the identification of the differences occurring between the proteomic patterns of the two investigated cell lines and the evaluation of the effect of the drug Trichostatin A on the protein content of the cells. PCA turned out to be a successful tool for the identification of the classes of samples present in the dataset. Moreover, the loadings analysis allowed the identification of the differentially expressed spots, which characterise each group of samples. The treatment of both the cell lines with Trichostatin A therefore showed an appreciable effect on the proteomic pattern of the treated samples. Identification of some of the most relevant spots was also performed by mass spectrometry.

Analysis of Variance↗

PubMed2004

Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes.

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules.

Adult↗

PubMed2004

Determination of the isoelectric point of proteins by capillary isoelectric focusing.

Different ways of determining isoelectric points (pI) of proteins in capillary isoelectric focusing are reviewed here. Due to the impossibility of direct pH measurements in the liquid phase, such assessments have to rely on the use of pI markers. Different types of pI markers have been described: dyes, fluorescently labelled peptides, sets of proteins of known pI values. It appears that, perhaps, the best system is a set of 16 synthetic peptides, trimers to hexamers, made to contain each a Trp residue for easy detection at 280 nm. By a careful blend of acidic (Asp, Glu), mildly basic, with pK around neutrality (His), and basic (Lys, Arg) amino acids, it is possible to obtain a series of pI markers with pI values quite evenly distributed along the pH scale, possessing good buffering capacity and conductivity around their pI values and thus focusing as sharp peaks. Another approach to pI determination is the monitoring of the current during mobilization: this allows, with the aid of known pI markers, to calibrate the system with a pI/current graph. Pitfalls and common errors in pI determinations are reviewed here and guidelines given for minimizing such errors in pI estimation.

Chemical Phenomena↗

PubMed2004

Properties of poly-aminophenylboronate coatings in capillary electrophoresis for the selective separation of diastereoisomers and glycoproteins.

The polymerisation of 3-aminophenylboronic acid (APBA) in aqueous environment has been used for the open tubular modification of capillary electrophoresis (CE) capillaries. Being poly-APBA endowed with boronic acid, aromatic rings and secondary amines groups, it posses a variety of functional groups affecting selectivity. Diastereoisomers (e.g. ascorbic and isoascorbic acid) and proteins (e.g. haemoglobins) were successfully separated onto poly-APBA column, by means of a combination of electrophoresis and open tubular electrochromatography. The mechanism of selection was investigated: results indicate an interplay between enhancing or silencing the contribution of the protonable functionalities (amino groups, boronic acid). The properties of APBA polymer coating make it attractive for CE separation and for further application in affinity separations and chip technologies.

Boronic Acids↗

PubMed2004

Peptide and protein separations by capillary electrophoresis in the presence of mono- and diquaternarized diamines.

Two compounds, derivatives of 1,4-diazobicyclo[2,2,2]octane (DABCO), have been evaluated as potential quenchers of silanol interactions with peptides and proteins during their capillary zone electrophoresis (CZE) separations. They are: 1-(4-iodobutyl)4-aza-1-azoniabicyclo[2,2,2]octane iodide (M7C4I) and 1,4-didecyl-1,4-diazoniabicyclo [2,2,2]octane dibromide (C10M7C10). The first compound is known to react with the wall, by forming a covalent bond via alkylation of silanols. On the contrary, the second one (C10M7C10) can only loosely interact with silica due to lack of reactive iodine and to a much too short distance (a C(2)) between the two quaternary nitrogens. Very good peptide maps of protein digests can be obtained in isoelectric glutamic acid (Glu) buffer, at pH 3.52 by utilizing the M7C4I. However, in the case of total tissue extracts, excellent resolution is obtained only with the first eluting part of the analyte spectrum (i.e., peptides and smaller proteins). With larger proteins, interaction with the wall and loss of resolution is experienced. When using the C10M7C10, good resolution of di- and tripeptides is obtained, while a loss of resolution is observed with entire protein digest. M7C4I does not seem to interact with the peptide/protein analytes, and simply repels them from the wall via its positive charges; it is believed that disalt (C10M7C10) acts by interacting with the same compounds, possibly by forming micelles in solution.

Animals↗

PubMed2004

Bioanalysis: its past, present, and some future.

An overview of about 100 years of bioanalysis is here disastrously attempted. The beginning of rigorous analytical systems can perhaps be traced back to the building and testing of the analytical ultracentrifuge by Svedberg and the apparatus for moving-boundary electrophoresis of Tiselius, both systems relying on expensive and hard to operate machines. In the sixties, Porath discovered porous beads for the determination of relative molecular mass (Mr) of proteins, based on the principle of molecular sieving. Concomitantly, Svensson and his pupil Vesterberg described a revolutionary principle for fractionating proteins in a nonisocratic environment, based on generation of stable pH gradients in an electric field, a technique that went down to history as isoelectric focusing (IEF). Polyacrylamide gel electrophoresis (PAGE), with the brilliant idea of discontinuous buffers, was brought to the limelight and in 1967, sodium dodecyl sulfate (SDS)-PAGE was described, permitting easy assessment of protein purity and reasonable measurements of Mr values of denatured polypeptide chains. By the mid seventies, another explosive concept was realized: orthogonal combination of two unrelated techniques, based on surface charge and mass fractionation, namely, two-dimensional (2-D) PAGE already in the very first papers by O'Farrell elaborated to its utmost sophistication. The eighties saw the systematic growth of 2-D PAGE, accompanied by systematic efforts to develop instrumentation for large-scale production of 2-D maps and computer evaluation for 2-D map analysis, based on the sophisticated algorithms adopted by astronomers for mapping stars in the sky. Another fundamental innovation in the field of IEF was the discovery of immobilized pH gradients (IPGs) that brought the much needed reproducibility in 2-D maps while allowing exquisite resolution in very narrow pH ranges. The nineties were definitely the decade of capillary zone electrophoresis, with the concomitant concept of automation and miniaturization in electrokinetic methodologies. Also 2-D map analysis witnessed a big revival, thanks to the adoption of IPGs for the first dimension. The enormous progress of mass spectrometry resulted in first reports on the analysis of macromolecules and the building of data bases on gene and protein banks. The third millennium is, perhaps, exasperating the concept of miniaturization at all costs, while not disdaining increasingly larger maps for 2-D analysis of complex protein mixtures.

Animals↗

PubMed2004

Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.

A modified Neuhoff's colloidal Coomassie Blue G-250 stain is reported, dubbed "blue silver" on account of its considerably higher sensitivity, approaching the one of conventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The "blue silver" exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the "blue silver" exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of bovine serum albumin (BSA) gave a detection limit (signal-to-noise ratio > 3) of 1 ng in a single zone. The somewhat lower sensitivity of "blue silver" as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two-dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.

Animals↗