Use Of Immobilized pH Gradients Research Articles

Mining the oncoproteome and studying molecular interactions for biomarker development by 2DE, ChIP and SPR technologies

This article provides a comprehensive updated review of three proteomics technologies (2D gel electrophoresis [2DE]; chromatin immunoprecipitation [ChIP] and its related alternates, and surface plasmon resonance [SPR]), and their use in cancer biomarker discovery and studying molecular interactions. 2DE proteomics has an advantage in visualizing changes in the intact molecular weight and isoelectric point (pI) of a protein, which reflect biologically significant processing and charge alterations in addition to post-translation modifications. However, proteins that are hydrophobic, low abundance or with extreme pIs or molecular weight, are poorly represented. Despite these shortfalls, improvements have been made that provide enhanced resolution by use of immobilized pH gradients (IPGs), narrow strip large 2DE gels, and serial and parallel formats. Prefractionation of the sample is essential for studying low-abundance markers, although it subjects the experimental design to additional nonbiologic variations. 2DE has been applied to the successful identification of oncoproteins in serum and tissues of cancers, such as colon, breast, prostate and pancreas. Interactions between protein and DNA are essential for cellular function. The process of developing global approaches to studying chromatin began with the in vitro characterization of chromatin structural components by the versatile ChIP assay and its modifications, which are capable of analyzing protein–DNA interactions in vivo. These methods have been used to assess gene expression, transcription factor binding and/or histone modifications in various organisms, including human. Label-free, real-time analysis of interactions in biological systems employing SPR biosensors that measure changes in refractive index of solvents near the surface during complex formation or dissociation of proteins, oligonucleotides, oligosaccharides and lipids to small molecules, phage, viral particles and cells is used extensively in research laboratories and the pharmaceutical and biotechnology industries for studying drug discovery, biopharmaceutical developments and clinical immunogenicity assays, as well as in manufacturing and quality-control methods.

A comparison of immobilized pH gradient isoelectric focusing and strong‐cation‐exchange chromatography as a first dimension in shotgun proteomics

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.

Two‐Dimensional Gel Electrophoresis

While one-dimensional SDS-PAGE separates proteins on the basis of size, two-dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two-dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two-dimensional protein databases.

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.

Improved two-dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles by using immobilized pH gradients.

AbstractAn improved method for two‐dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles is presented. With the use of immobilized pH gradients in the first dimension, this method gave stable separations, with the proteins evently distributed over the entire gel surface. The number of spots in the electrophoretograms have increased fourfold compared to our earlier separations.

Immobilized pH gradients: Analytical and preparative use

The use of immobilized pH gradients for two-dimensional electrophoresis overcomes several of the limitations of carrier ampholyte-based isoelectric focusing. Procedures followed in the authors' laboratory for the analytical or preparative use of immobilized pH gradients are presented.

Highly resolving two-dimensional gels for protein sequencing.

Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort.

Orosomucoid (alpha-1 acid glycoprotein) phenotyping by use of immobilized pH gradients with 8 M urea and immunoblotting. A new variant encountered in a population study.

Orosomucoid (ORM) phenotyping has been performed on 329 unrelated Swiss subjects, using immobilized pH gradients with 8 M urea and 2% v/v 2-mercaptoethanol followed by immunoblotting. After desialylation the band patterns of ORM confirmed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles (ORM1*F1, ORM1*S and ORM1*F2). One rare and one new allele were detected. The rare variant, tentatively assigned to the second structural locus ORM2, is observed in a cathodal position and named ORM2 B1. The new variant, tentatively assigned to the first structural locus ORM1, is observed in a region located between ORM1 S and ORM1 F2, and named ORM1 F3. Moreover, the pI values of the ORM variants have been measured accurately with Immobiline Dry Plates (LKB): they were found to be within the pH range 4.93-5.14.

Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.

This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.

High resolution two‐dimensional polyacrylamide gel electrophoresis of basic myeloid polypeptides: Use of immobilized pH gradients in the first dimension

AbstractA two‐dimensional polyacrylamide gel electrophoresis approach for the separation of urea/detergent solubilized basic proteins is presented. An immobilized pH gradient (IPG) gel, pH range 7–10, containing 8 M urea, 2% Nonidet P‐40 and 10 mM dithioerythritol was used for the first‐dimensional isoelectric focusing separation. Approximately 400 to 500 basic polypeptides from myeloblasts or neutrophils were visualized with silver staining. The IPG gel in some cases included carrier ampholytes in the same pH range. The carrier ampholyte‐modified (“hybrid”) IPG gel exhibited improved resolution of some polypeptides, but at the expense of reproducibility in the focusing of other polypeptides. Transfer of sample into the IPG gel was facilitated by a combination of low initial voltage, presence of carrier ampholytes in the sample and application of the sample to the surface of the gel within a containment collar. 6 M urea was required in the SDS equilibration solution for the efficient transfer and high resolution separation of polypeptides in the second‐dimensional sodium dodecyl sulfate‐gel. Absolute spot position was highly reproducible among gels prepared simultaneously. Standard deviations of ± 0.84 mm for the focusing and ± 1.25 mm for the molecular weight dimensions of 70 randomly selected spots were obtained. Integrated spot intensities ranged from 0.17 to 80 O.D. × mm2 with a median of 1.5 O.D. × mm2.