Immobilized pH Gradient Gels Research Articles

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox

BackgroundBothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.ResultsOptimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3–10 pH range during the isoelectric focusing step and 10–20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins.ConclusionThe analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

Immobilized pH Gradient Gel Cell To Study the pH Dependence of Drug Lipophilicity

An experimental setup to study the pH dependence of standard ion-transfer potentials at the water/NPOE interface is presented. The system is composed of a microhole generated by laser photoablation in a 12-microm polyethylene terephthalate film, the aqueous phase consisting of a commercial immobilized pH gradient gel reswelled in electrolyte solution and a droplet of organic solution. Two electrodes are used, an Ag/AgCl aqueous electrode and an Ag/AgTPBCl organic electrode. This setup is applied to the study of two ionizable compounds (pyridine, 2,4-dinitrophenol). Thermodynamic parameters such as the standard transfer potential, the Gibbs energy of transfer, and the partition coefficients of the ionized forms as well as the neutral forms of these drug compounds are evaluated by differential pulse voltammetry. The data obtained are summarized in ionic partition diagrams, which are a useful tool for predicting and interpreting the transfer behavior of ionized drugs at the liquid/liquid interfaces mimicking the biological membranes.

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.

Differential Radioactive Proteomic Analysis of Microdissected Renal Cell Carcinoma Tissue by 54 cm Isoelectric Focusing in Serial Immobilized pH Gradient Gels

We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 microg renal cell carcinoma protein and 3.8 microg normal kidney protein respectively from one patient. The study involved separate radio-iodination of each sample with both (125)I and (131)I, dual inverse replicate sample loading to high resolution 54 cm "daisy chain" serial immobilized pH gradient isoelectric focusing (IPG-IEF) 2D-PAGE gels, co-electrophoretic separation of cross-labeled proteins from different samples, and precision multiplex differential radioactive imaging to obtain signals specific for each sample coelectrophoresed within single gels but labeled with different isotopes of iodine, providing extremely precise intra-gel estimates of the abundance ratio for protein spots from both samples. Twelve multiplexed analytical radioactive SDS-gels from 4 serial IPG-IEF gels provided 24 individual radioactive images for a comprehensive analytical protein multiplex quantification study. A further 12 SDS gels containing (125)I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed <40% of the (125)I-labeled sample, and <20% of the (131)I-labeled sample from the respective original 3.8 microg samples. Twenty-nine proteins were identified by mass spectrometry with PMF scores >70 that were >2-fold differentially abundant between the samples and t-test probabilities <0.05. We conclude that this combination of technologies provides excellent quality protein multiplex data for the differential abundance analysis of large numbers of proteins from extremely small samples, and is applicable to a broad range of clinical and related applications.

Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis

We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271). Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term "daisy chain IEF". Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (approximately 100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12.

Salt removal during Off-Gel? electrophoresis of protein samples

The Off-Gel technology was recently described for protein fractionation in a solution placed on top of an immobilized pH gradient gel. In addition, this process was found to remove salts from the biological samples to analyze. This desalting effect is studied experimentally in a conductometric prototype cell. A simplified analytical model is developed to understand this process and a good agreement is found with the conductivity measurements. To illustrate the desalting of a biological sample, a 1 mg.mL(-1) solution of beta-lactoglobulin A in 0.1 M NaCl is subjected to electrophoresis in a single compartment Off-Gel cell. The analysis of the resulting sample by ESI-MS demonstrates the effective removal of salt. A finite element diffusion-migration model is also used to illustrate how the nonuniformity of the electric field in the cell, associated with the salt migration, can slow down the desalting process.

Analysis of high-density lipoprotein apolipoproteins recovered from specific immobilized pH gradient gel pI domains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The proteins associated with the circulating lipoproteins in the blood function not only for mediating lipid metabolism but also for maintaining structural stability of the micellelike structure. Any modifications of these proteins, by mutation or posttranslational modification, could compromise the function of these proteins and contribute to the development of cardiovascular disease. Because of the presence of extensive lipophilic domains, these proteins, when recovered from the lipoprotein particle (apolipoproteins) present an analytical challenge because of low solubility and proclivity toward aggregate formation. Our goal is to characterize these proteins by a combination of high-accuracy pI measurement coupled with MALDI analysis. In this report, we demonstrate the high resolution of immobilized pH gradient isoelectric focusing (IPG-IEF) for the analysis of these apolipoproteins isolated from serum HDL collected from a density gradient ultracentrifugation-based separation. The IPG separation of the HDL apolipoproteins was imaged and combined with digital analysis to produce a detailed pI profile of the apolipoproteins in the high-density lipoprotein (HDL) fraction. This is the first time that a high-resolution pI profile has been obtained for the HDL apolipoproteins. The feasibility of linking the pI profile to a MALDI-based molecular weight determination was achieved by incorporating passive elution of the intact proteins from the IPG gel with a four-component solvent mixture that solved the problem of recovery of the apolipoproteins from the IPG matrix. Twenty-five bands were observed in the pI profile. A survey analysis of 12 of these bands by MALDI indicated that they were associated with the known HDL apolipoproteins. While there is considerable overlap in the pI profiles of the apolipoproteins, linking the analysis with a MALDI-based second dimension in m/z is shown to be an efficient method to characterize this complex mixture of apolipoproteins.

Soft immobilized pH gradient gels in proteome analysis: a follow-up.

As a follow-up of a previous work on two-dimensional map analysis utilizing soft (< 4%T) immobilized pH gradient (IPG) matrices in the first dimension (Candiano et al., Electrophoresis 2002, 23, 292-297), we have further optimized the preparation of such dilute IPG gels. One important step for obtaining an even reswelling of the entire IPG strip along the pH 3-10 interval is a washing step in 100 mM citric acid. It appears as though after rinsing off the excess acid in distilled water, a gradient of this tricarboxylic acid remains trapped into the IPG matrix, from almost nil at the acidic gel region to substantially higher amounts in its basic counterpart. This gradient helps in obtaining a uniform reswelling of the IPG strip, since carboxyl groups are more heavily hydrated than amino groups. The combined effects of uniform reswelling and of diluting the gel matrix favor penetration of large macromolecules (> 200 kDa) and allow for better spot resolution and for the display of a substantially higher number of spots also in the 30-60 000 Da region. A delipidation step in tri-n-butylphosphate:acetone:methanol (1:12:1) appears to substantially improve spot focusing and greatly diminish streaking and smearing of spots in all regions of the pH gradient.

Isoelectric focusing in long immobilized pH gradient gels to improve protein separation in proteomic analysis.

The number of protein spots detected on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) gels increases as the gel size increases. The largest commercially available systems resolve a few thousand spots, being only a fraction of the total proteome. We have developed an extremely long isoelectric focusing (IEF) system aimed at more complete protein profiling. The system is especially well suited to sensitive detection methods, such as radioactive detection. The major constraint preventing progress in this area has been the inability to create an even density gradient during the immobilized pH gradient (IPG) casting process. We demonstrate for the first time that this constraint can be effectively overcome, to enable greatly increased IEF separating power with all the advantages of IPG technology,

Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry.

Over the past several years, a large effort has been focused on improvements of two-dimensional (2-D) gel electrophoresis-based proteomics technology, and on development of novel approaches for proteome analysis. Here, we describe the application of an alternative strategy for the analysis of complex proteomes. The strategy combines isoelectric focusing in immobilized pH gradient strips (in-gel IEF), mass spectrometry (MS), and bioinformatics. A protein mixture is separated by in-gel IEF, and the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the tryptic peptides are subjected to liquid chromatography-nanoelectrospray-quadrupole ion-trap tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS data are used to identify the proteins through searches of a protein sequence database. Using this in-gel IEF-LC-MS/MS strategy, we have identified 127 proteins from a human pituitary. This study demonstrates the potential of the in-gel IEF-LC-MS/MS approach for analyses of complex mammalian proteomes.

Revisiting electroblotting of immobilized pH gradient gels: a new protocol for studying post-translational modification of proteins.

Currently, one of the most important techniques in proteome analysis is two-dimensional electrophoresis that is widely used for separation of thousands of different protein spots. Nevertheless, characterization of special aspects in protein patterns, e.g., separation of protein isoforms generated by post-translational modifications, requires individual detection methods, e.g., immunoblotting. Blotting of proteins after fractionation in immobilized pH gradients has always caused some problems. In this paper we present an optimized protocol for immunoblotting after isoelectric focusing using immobilized pH gradient (IPG) strips cast on Net-Fix as an internal support that is permeable to electric current. The focusing procedure can be carried out in commonly used IPG systems, e.g., the IPGphor by Amersham Biosciences, where electrically assisted rehydration can be performed. This may be of interest for many laboratories, because the same system as used for the first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is involved. As an example, we describe separation and detection of up to seven isoforms of recombinant erythropoietin beta using semidry blotting of IPG strips and visualization by chemiluminescence detection.

Two-dimensional maps in soft immobilized pH gradient gels: a new approach to the proteome of the Third Millennium.

Same major improvements in proteome analysis of cytosolic and membrane proteins by two-dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)-gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2-3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri-n-butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240-220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essentially none when delipidation was omitted. The present report also confirms the importance of a reduction and alkylation step of the protein sample prior to all electrophoretic steps, including focusing in the Immobiline gel, as recently reported by Herbert et al.

Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis

Comprehensive analysis of complex proteomes requires prefractionation of samples prior to two-dimensional gel electrophoresis (2-DE). This study demonstrates the utility of using a high resolution sample prefractionation method and slightly overlapping narrow pH range two-dimensional gel electrophoresis to enhance quantitative comparisons of complex proteomes. A key feature of this strategy is to prefractionate samples into a few well-defined pools using microscale solution isoelectric focusing (nusol-IEF) prior to 2-DE protein analysis. Sample prefractionation is achieved using a series of tandem small volume chambers (500 nuL) separated by thin membranes containing immobilines at specific pH's. The resulting well-resolved fractionated samples are optimally separated on a series of slightly overlapping narrow pH range immobilized pH gradient (IPG) gels, which are approximately 0.1 pH units wider than the nusol-IEF fractionated pools. When nusol-IEF prefractionation was applied to proteome analyses of mouse serum, it resulted in the capacity to separate much higher protein loads on narrow pH range IPG gels while retaining good resolution and spot recovery. More importantly, the prefractionation of serum greatly enhanced the ability to detect low abundance proteins, because major interfering proteins were removed from most fractions. At least 6- to 30-fold higher protein loads were possible for nonalbumin fractions on narrow pH range IPG gels. The dynamic range of protein detection is substantially increased since higher protein loads can be applied to narrow pH range 2-DE gels, and duplicate gels can be stained with colloidal Coomassie and silver stains for quantitation of abundant and minor proteins, respectively. Finally, the ability to effectively fractionate complex proteomes into very narrow ranges (< 0.5 pH units) strongly suggests that nusol-IEF could be used to prefractionate complex samples for subsequent direct analysis by liquid chromatography-tandem mass spectrometry methods as an alternative to using overlapping narrow pH range 2-DE gels.

Interaction of the SH2 domain of Fyn with a cytoskeletal protein, beta-adducin.

Fyn is a Src family tyrosine kinase expressed abundantly in neurons and believed to have specific functions in the brain. To understand the function of Fyn tyrosine kinase, we attempted to identify Fyn Src homology 2 (SH2) domain-binding proteins from a Nonidet P-40-insoluble fraction of the mouse brain. beta-Adducin, an actin filament-associated cytoskeletal protein, was isolated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. beta-Adducin was tyrosine phosphorylated by coexpression with wild type but not with a kinase-negative form of Fyn in COS-7 cells. Cell staining analysis showed that coexpression of beta-adducin with Fyn induced translocation of beta-adducin from the cytoplasm to the periphery of the cells where it was colocalized with actin filaments and Fyn. These findings suggest that tyrosine-phosphorylated beta-adducin associates with the SH2 domain of Fyn and colocalizes under plasma membranes.

An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis.

A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA-IEF) or immobilized pH gradient (IPG) gels by in-gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole-Sodium dodecyl sulfate (SDS)-zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry-compatible silver staining, but simpler and faster. Proteins were digested in imidazole-SDS-zinc stained CA-IEF and IPG gels in the presence of a zinc-chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high-sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.

Separation of Tubulin Isoforms by Isoelectric Focusing in Immobilized pH Gradient Gels

Separation of Tubulin Isoforms by Isoelectric Focusing in Immobilized pH Gradient Gels

Modified immobilized pH gradient gel strip equilibration procedure in SWISS-2DPAGE protocols.

In the present paper we report a revised protocol for immobilized pH gradient (IPG) gel strip equilibration involving a procedural modification between the first- and second-dimensional separation in both analytical and preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). By changing the pH of the equilibration buffer (pH 8.0), the concentration of alkylating reagent (125 mM iodoacetamide) and the time of incubation (15 min), it has been possible to achieve increased cysteine (Cys) alkylation to completion with only one adduct of carboxyamidomethyl-Cys formed. Importantly, the modification does not alter the 2-D proteome patterns and therefore maintains the integrity of the existing SWISS-2DPAGE entries. Results are presented for comparative analyses using human plasma, and for Cys analysis of human albumin to illustrate the advantages of the improved protein reduction and Cys alkylation. The modified step of IPG gel strip equilibration will assist protein digestion for matrix-assisted laser desorption/ionisation - time-of-flight - mass spectrometry analysis, and make Cys quantitation possible without further in-gel or on-blot alkylation.

Casting immobilized pH gradients (IPGs).

AbstractOne of the main requirements for a 2-D protocol is reproducibility of spot position, and, indeed, the technique of isoelectric focusing on immobilized pH gradients (IPGs) is ideally suited to provide highly reproducible 1-d separations. IPGs are obtained through the copolymerization of acidic and basic acrylamido derivatives of different pKs within a polyacrylamide matrix (1,2) (Fig. 1). The pH gradient may be devised by computer modeling either with a linear or with an exponential course. IPGs are cast from two limiting solutions containing the buffering chemicals at concentrations adjusted to give the required pH course upon linear mixing. For consistent results, gradient pouring and polymerization are carried out under controlled conditions. The covalent nature of the chemical bonds formed during the polymerization step results in a permanent stability of the pH gradient within the matrix. Conflicting requirements during the focusing procedure prevent any effective use of IPGs into capillary tubes (3): The need to buffer with carrier ampholytes (CAs) the pH extremes caused by the migration of the polymerization catalysts is contrasted by the adverse effects of the electroendosmotic flow brought about by the addition of CAs to the gel phase. The demand for the IPG gels to be backed by a binding support—they are usually cast on GelBond™ foils—results in dimensional stability between 1-d and 2-d as a further assistance to reproducibility. Structure of the IPG matrix. Different regions of an IPG made up with a basic and an acidic Immobiline are schematically drawn from cathode to anode. In each region, pH depends from the concentration ratio between the basic and the acidic dissociating groups. From the Henderson-Hasselbalch equation, where the concentration of the basic monomer is twice the concentration of the acidic monomer, pH equals the pK of the base, where the concentration of the basic monomer is one-half the concentration of the acidic monomer pH equals pK of the acid, and where the two concentrations are alike, pH equals the mean between the two pKs. KeywordsOutlet TubingPolymerization StepRubber GasketCarrier AmpholyteMolding PlateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Salivary electrophoresis in the diagnosis of Sjögren's syndrome

Objective. The purpose of this study was to investigate the potential use of salivary electrophoresis for the diagnosis of Sjögren's syndrome. Methods. Salivary protein profiles of 43 patients and 39 healthy control subjects were compared on three different gel electrophoresis systems: sodium dodecylsulfate-polyacrylamide gel electrophoresis, anionic polyacrylamide gel electrophoresis, and immobilized pH gradient gel electrophoresis (isoelectric point, 3.5−5.0). Results. Most of the patients with Sjögren's syndrome exhibited an electrophoretic profile that was different from that of the healthy control subjects. Among the three gel electrophoresis systems examined, the immobilized pH gradient system appeared to be the most reliable for Sjögren's syndrome. Tests of accuracy revealed that the immobilized pH gradient system exhibits high specificity (97%), sensitivity (95%), positive predictive value (97%), and negative predictive value (95%) in the diagnosis of Sjögren's syndrome. Conclusions. Our results suggest that salivary electrophoresis is a potentially useful test for the diagnosis of Sjögren's syndrome.

Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients.

A simple and inexpensive methacrylate rehydration chamber was built to accommodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 microL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.