Immobilized pH gradients (IPG) simulator--an additional step in pH gradient engineering: I. Linear pH gradients.

A new computer program, called immobilized pH gradients (IPG) simulator, is proposed for calculating and optimizing any recipe for use in isoelectric focusing in immobilized pH gradients. Unlike our previous monoprotic electrolyte gradient simulation (MGS) and polyelectrolyte gradient simulation (PGS) programs, based on minimizing CV(beta), the present program has a target function the minimization of the quadratic moment around zero of the residuals (mu 2). With this algorithm it is possible to formulate IPG recipes which have deviations from linearity well below 1% of the given pH interval (a limit set with the previous MGS and PGS programs), in fact, as small as 0.1-0.2% (in pH units). The new simulator performs 2-3 times better than the previous ones in the pH 4-10 range, and is absolutely necessary when working outside this range, at extreme pH values, where CV(beta) cannot work against the buffering power of bulk water, thus generating pH recipes with huge deviations from linearity. In the latter cases, mu 2 performs 10 times better than CV(beta). When utilizing strong titrants for extended pH intervals, the "all or none" rule has been discovered: such titrants should always be used in tandem, since omission of one of the two at either the acidic or basic extremes produces strongly distorted pH profiles. Our new, most powerful simulator also contains equations for creating nonlinear gradients, notably: concave and convex exponentials and sigmoidal (see the companion paper: Righetti, P. G. and Tonani, C., Electrophoresis 1991, 12, 1021-1027).     

Immobilized pH 2.5-11 gradients for two-dimensional electrophoresis.

Extremely wide immobilized pH gradients, pH 2.5-11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two-dimensional map.     

Immobilized pH gradients in ultrathin-layer (100 micron) gels: avoiding capillarity by a 'squeezing-sealing mold' technique

Several procedures for casting 100 microns ultrathin immobilized pH gradients are described. When acrylate/glass molding cassettes (Pascali et al., Electrophoresis 1987, 8, 371-373) are used two main problems are encountered: (i) a tendency of polymerization solutions, at the beginning of delivery, to spread across the glass surfaces with troublesome effects on the gradient stratification, and (ii) the raising of steep menisci at both extremities of the pH intervals, originating from capillarity phenomena and resulting in nonuniform gradients with bowed electrophoretic patterns. The first shortcoming was acceptably solved by increasing the density of sucrose gradients, and pouring them into prewarmed molding cassettes. The detrimental effect of menisci could be overcome by using a 'squeezing-sealing mold' technique. A molding cassette was endowed with a continuous, squared spacer frame, the upper side being open by inserting a wedged clip. A slight excess of polymerization solution was first dispensed into the cassette and squeezed away on removal of the clip. By completely excluding air from the molding cassette, uniform and well reproducible ultrathin gels could be cast. A major advantage of ultrathin immobilized pH gradient gels is the drastically shorter focusing time.     

Immobilized pH gradients: effect of salts, added carrier ampholytes and voltage gradients on protein patterns

Salts formed from strong acids and bases (e.g. NaCl, Na2SO4, Na2HPO4), present in a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (greater than 50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of "strong" salts in the sample zone with salts formed by weak acids and bases, e.g.. Tris-acetate, Tris-glycinate, Good's buffers such as (N-[2-acetamido]-2-iminodiacetic acid (ADA), (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid (ACES), (3-[N-morpholino]propane sulfonic acid (MOPS), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of "strong" salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e.g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high-voltage settings). Guidelines are given for trouble-free IPG operations.     

Nonamphoteric isoelectric focusing: II. Stability of borate-glycerol pH gradients in recycling isoelectric focusing

By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio of polyol to borate over the useful pH range of 4.0-6.0, it is possible to control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethylamino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (pI4.70) migrated to the pH 4.61 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

Barley cultivar discrimination: II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients.

Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol-soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2-11 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitol-soluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.     

The adsorption of large proteins in electrofocusing on immobilized pH gradients: II. Dependence on the oligomeric state of Immobiline

Five proteins with molecular mass in excess of 200 kDa were found to adsorb onto gels during isoelectric focusing on immobilized pH gradients (IPGEF). To probe for the mechanism of that adsorption, the homogeneity of the six Immobiline preparations used to make IPGEF gels was tested. Five of these Immobiline preparations appear homogeneous in gel filtration of Sephadex G-10. The sixth Immobiline (pK 9.3) exhibits a minor component eluting ahead of the major peak and comprising less than 4% of the total Immobiline absorbing at 226 nm. The proportion of the minor component increases with column load. Major and minor components when isolated appear to equilibrate with one another. Judging by the results of mass spectrometry, all 6 preparations are free of small aggregates of less than 500-600 Da molecular mass. Ultrafiltration of the Immobiline preparations through a membrane with 500 Da nominal cutoff leads to partial desorption of only 3 of the 5 adsorbed proteins. CHAPS is ineffectual in desorbing the 5 proteins from the IPG gel made with ultrafiltered Immobilines. None of the 6 Immobiline preparations used precipitates ferritin. All large proteins that adsorb onto IPGEF gels in the pH range 4-9.5 also adsorb onto commercial IPGEF gels in the pH range 4-7.     

Electrocatalytic Activity of Immobilized Co(II) on Porous Graphene Aerogels

The highly‐porous graphene aerogel (GA) with BET surface area of 810 m² g^?1 and three‐dimensional structure has been successfully fabricated using the hydrothermal technique. The modified glassy carbon electrode was then prepared by casting the graphene aerogel solution followed by immersing the GA/GCE in Co⁺² solution. The results showed that graphene aerogels improved the adsorption ability of the Co (II) ions. The electrocatalysis of oxygen evolution reaction (OER) at the Co‐GA modified glassy carbon elec‐ trode (Co‐GA/GCE) has been investigated using linear sweep voltammetry (LSV) in alkaline solution. The OER was noticeably enhanced at Co‐GA/GCE, representing a negative shift in the LSV curve at the Co‐GA/GCE compared to that obtained at the bare GCE. The high electrocatalytic activity, good reproducibility and low cost of proposed electrode provides desired characteristics of a potential candi‐ date in the industrial water electrolysis process.     

Two‐step Immobilized Metal Affinity Chromatography (IMAC) for Phosphoproteomics Using Mass Spectrometry

In this study, a new strategy named two‐step IMAC is demonstrated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a protein mixture. In this method, the first IMAC was performed at the protein level to extract the minute amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was performed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI‐MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β‐casein that was spiked in a protein mixture can be successfully extracted by the first IMAC at a concentration of less than 1–3%, and the two phosphopeptides of β‐casein with single and four phosphorylation sites, respectively, can be captured by the second IMAC. It was found that the two‐step IMAC method could significantly reduce non‐specific bindings from unwanted proteins and greatly enhance the MALDI‐MS signal of phosphopeptide ions compared to the typical one‐step IMAC, by which only IMAC at the peptide level was performed. Two‐step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one‐step IMAC, which is especially important when analyzing complicated unknown samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be degraded by the presence of biological matrixes, such as the cell lysate in which the β‐casein was spiked in.     

Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides

In this work, we demonstrate the potential use of immobilized pH gradient isoelectric focusing as a first dimension in shotgun proteomics. The high resolving power and resulting reduction in matrix ionization effects due to analyzing peptides with almost the exact same physiochemical properties, represents a significant improvement in performance over traditional strong cation-exchange first-dimensional analysis associated with the shotgun proteomics approach. For example, using this technology, we were able to identify more than 6000 peptides and > 1200 proteins from the cytosolic fraction of Escherichia coli from approximately 10 microg of material analyzed in the second-dimensional liquid chromatography-tandem mass spectrometry experiment. Sample loads on the order of 1 mg can be resolved to 0.25 isoelectric point (pI) units, which make it possible to analyze organisms with significantly larger genomes/proteomes. Accurate pI prediction can then be employed using currently available algorithms to very effectively filter data for peptide/protein identification, and thus lowering the false-positive rate for cross-correlation-based peptide identification algorithms. By simplifying the protein mixture problem to tryptic peptides, the effect of specific amino acids on pI prediction can be evaluated as a function of their position in the peptide chain.     

High-performance chromatofocusing using linear and concave pH gradients formed with simple buffer mixtures. II. Separation of proteins

The separation of proteins using high-performance chromatofocusing with linear or concave pH gradients formed using simple mixtures of buffering species in the elution buffer is investigated experimentally. The separation achieved is comparable to that using polyampholyte elution buffers with these types of systems. More specifically, protein band widths at one half of the band height in the range between 0.1 and 0.025 pH units were observed, and good resolution was achieved of protein variants differing by a single amino acid residue in separation times of 30 min or less. An especially useful elution buffer is investigated that contains only four buffering species and that produces a linear pH gradient in the range between pH 9.5 and 6.0 when used together with a particular high-performance column packing made specifically for chromatofocusing. This elution buffer and column packing combination is evaluated by using it for the chromatofocusing of equine myoglobin and human hemoglobin variants. Additional applications are described in which a polyethyleneimine derivatized silica column packing and a pH gradient that is concave in shape are used for the separation of proteins in an E. coli cell lysate.     

Density gradients in packed columns: II. Effects of density gradients on efficiency in supercritical fluid separations

To investigate how fluid compressibility affects efficiency in supercritical fluid separations, band dispersion along a packed capillary column was measured from on-column elution rate profiles obtained under solvating gas chromatography (SGC) conditions; this allowed efficiency to be determined with respect to position along the column. Theoretical efficiency was also modeled. The model indicates that the primary cause of band broadening in SGC is high mobile phase velocity near the column outlet. However, the experimental results show that significant band broadening also occurs near the column inlet in a region that corresponds to high elution rates of the analyte. On-column detection also revealed spatial focusing of the analyte as it moves down the column density gradient.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

Preparation of gradient copolymers via ATRP in miniemulsion. II. Forced gradient

A series of forced gradient copolymers with different controlled distribution of monomer units along the copolymer backbone were successfully prepared by atom transfer radical polymerization in miniemulsion. The newly developed initiation technique, known as activators generated by electron transfer, was beneficial for forced gradient copolymers preparation because all polymer chains were initiated within the miniemulsion droplets and the miniemulsion remained stable throughout the entire polymerization. Various monomer pairs with different reactivity ratios were examined in this study, including n‐butyl acrylate/t‐butyl acrylate, n‐butyl methacrylate/methyl methacrylate, and n‐butyl acrylate/styrene. In each case, the added monomer diffused across the aqueous suspending medium and gradient copolymers with different forced distributions of comonomer units along the polymer backbone were obtained. The shape of the gradient along the backbone of the copolymers was influenced by the molar ratio of the monomers, the reactivity ratio of the comonomers as well as the feeding rate. The shape of the gradient was also affected by the relative hydrophobicities of the comonomers. Copolymerizations exhibited good control for all feeding rates and comonomer feeding ratios, as evidenced by narrow molecular weight distribution (M_w/M_n = 1.20–1.40) and molecular weight increasing smoothly with polymer yield, indicating high initiation efficiency. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 1413–1423, 2007     

1,8-dihydroxyanthraquinone-Mg(II) complex: II. Spectrophotometric study. Determination of Mg(II)

The 1,8-dihydroxyanthraquinone (1,8-DHAn) shows a colored and fluorescent reaction with the ion Mg(II) in a hydroalcoholic and ammonical medium.In the present work we have studied spectrophotometrically the 1,8-DHAn-Mg(II) complex in a hydroethanolic and ammonical, 8 × 10^?4M medium. We found that the complex shows a maximum absorbance at 510 nm, and obeys a 1:1 stoichiometry with log K of 4.08.We propose a new method for the spectrophotometric determination of Mg(II) which is valid for concentrations between 0.25 and 2.00 ppm, and yields an error of 1.32%.