Ionic equilibria in neutral amphiprotic solvents of low dielectric constant: Buffer solutions

The ionic equilibria in neutral amphiprotic solvents (isopropyl and tert-butyl alcohols) have been established, and equations to calculate pH values in solutions of acids, bases, salts or their mixtures, developed. The effect, on the dissociation equilibria, of the presence of small quantities of water or other solvents in the bulk solvent used has been taken into account in the proposed equations. On the basis of these equations some buffer solutions have been studied and recommended for electrode standardization. The results, tested by experimental work, show the importance of the incompleteness of dissociation of salts in these solvents, which decreases the pH of acid buffers and increases the buffer capacity.     

Trypsin digestion of proteins on intact immobilized pH gradient strips for surface matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

Isolelectric focusing (IEF) of proteins on immobilized pH gradient (IPG) strips is an integral part of two-dimensional (2D) electrophoresis-based proteomics. Proteins can be effectively analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) on the intact strip itself, leading to the creation of a virtual 2D map giving pI and MW information, bypassing the second dimension SDS-PAGE. Further, trypsin digestion of proteins on the strip can significantly aid the identification of IPG-separated proteins. However, the small size of the peptides leads to diffusion along and outside the gel matrix. In this study, we describe a simple spray-based procedure to perform 'on-strip' trypsin digestion of proteins embedded in IPG strips. Examination of intact myoglobin and its tryptic peptides shows that post-digestion diffusion of tryptic peptides is significantly minimized using this approach.     

Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid phosphatase typing for breeding nematode-resistant tomatoes by isoelectric focusing with an ultranarrow immobilized pH gradient

The genetic variants of tomato acid phosphatase (Aps-1) systems have been analyzed by isoelectric focusing in immobilized pH gradients (IPG). By using an ultranarrow pH 4.25-4.55 IPG gel, the two genotypes Aps-1(1) and Aps-1+, differentiating tomato variants into nematode-resistant or nematode-susceptible plants, are separated into two sharp zones over a distance of 2.5 cm with isoelectric points of 4.37 and 4.43, respectively. Under these conditions, silver staining of the Aps-1 variants proved to be superior to enzyme staining. By applying more than 50 samples on one IPG gel, this method proved to be a powerful tool for reliable tomato nematode resistance screening.     

Two-dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): method, reproducibility and genetic aspects

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.     

Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers

Nonaqueous capillary electrophoresis was evaluated for its potential to separate peptides in methanolic background electrolytes in comparison to aqueous-methanol (50% v/v) and water. Isomeric aspartyl dipeptides and Leu- and Met-enkephalin served as model compounds. pK(a) values were determined in the three solvent systems based on the apparent pH scale and in the case of methanol additionally based on the conventional pH scale. Changing from water to methanol led to an increase of the ionization constants describing the dissociation equilibria of the carboxyl group and the amino group, respectively. The pK(a) shift was more pronounced for the carboxylic acid function leading to a compression of the mobility-pH curve. As reported for aqueous buffers, efficient separations of the peptides were achieved in methanolic background electrolytes including the resolution of the diastereomers of the isomeric alpha- and beta-aspartyl dipeptides. In contrast to aqueous buffers, the separation of Leu- and Met-enkephalin could also be obtained in buffers in methanol at high pH.     

Two-dimensional gel electrophoresis using immobilized pH gradient tube gels

An apparatus for the preparation of gels for immobilized pH gradient isoelectric focusing (IPG) in glass tubes was developed. Using this apparatus, the highly reproducible immobilized pH gradient can be formed with Immobilines in polyacrylamide gels, and IPG gels at all possible pH ranges can be easily prepared at low cost. The IPG tube gels in the first dimension in two-dimensional gel electrophoresis was used to separate and identify a number of rice embryo proteins in the proteome analysis. There was no difference in resolution of proteins between the tube gels and the commercially available slab gels; after electrophoresis, however, we could efficiently obtain a larger amount of the purified proteins from the tube gels than from the slab gels.     

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

In the presence of methanol and n‐decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3–10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M‐IPG). In addition, an online self‐built platform for protein separation was established on account of the introduction of a cross‐shaped unit and two short‐off valves. In this platform, a cross‐shaped unit was not only used to join the M‐IPG column and a six‐way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short‐off valve in the tee unit or cross‐shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7‐proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M‐IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A_1c have been successfully resolved with the newly developed M‐IPG column.     

Two-dimensional gel electrophoresis of platelet polypeptides with immobilized pH gradients in capillary tubes

Two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG) gels in capillary tubes was used in the first-dimensional isoelectric focusing (IEF) for the separation of human platelet polypeptides. Two types of IPG tube gels, pH ranges 4-8 and 7-10, containing 8 M urea, 1% Nonidet P-40 and 0.1% pH 3.5-10 Ampholine carrier ampholytes (CA) were prepared by a simple method not requiring special equipment. The addition of CA to both gel and sample solutions was essential in the tube gel IPG system. Proteins were visualized by a modification of Wray's silver-staining technique. The degree of resolution and the number of spots observed on an IPG 2-DE gel with pH 4-8 were comparable with those obtained with O'Farrell's high-resolution 2-DE. Approximately 200 basic polypeptides, which are difficult to separate by conventional CA-based IEF 2-DE or the non-equilibrium pH gradient system, were well resolved by 2-DE with a pH 7-10 IPG tube gel in the first-dimension. The gel patterns with either pH gradient 4-8 or 7-10 were highly reproducible among gels prepared and run simultaneously. These results demonstrated the potential and usefulness of the 2-DE system with IPG gels in capillary tubes.     

The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes

The last few years have brought dramatic improvements for sample preparation and solubilization of protein for electrophoretic analyses. The use of reagents such as thiourea and novel sulfobetaine surfactants increases the total number of proteins able to be visualized from a complex mixture such as a cell lysate and also allows more hydrophobic membrane proteins to be resolved. As the red blood cell (RBC) contain no organelles, it is an ideal source of relatively pure plasma membrane for protein solubilization studies. In addition, there are a number of diseases related to abnormalities of RBCs proteins, thus it is of medical relevance as well as a test sample for technology development. However, the procedure for purifying RBC membranes is rather time-consuming and is normally carried out under almost physiological conditions, which can be conducive to proteolytic degradation of the membrane proteins. Significant differences in two-dimensional (2-D) patterns with and without protease inhibitors in sample preparation are demonstrated. In addition, is shown that preparation of RBC membranes with sodium carbonate, pH 11, leads to multimeric complexes of hemoglobin and causes hemoglobin to be irreversibly attached to the membrane. When using immobilized pH gradients (IPG) as the first dimension, it is demonstrated that the spectrins (large, filamentous proteins of 280 kDa) are lost from the 2-D map unless active, instead of passive, sample hydration into the IPG strip is adopted.     

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.     

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.     

Isoenzyme analysis of lichen algae in immobilized pH gradients

A base for a modern species' concept of chlorococcal algae can be obtained not by morphological analysis, but by biochemical characters, e.g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4-7 IPG interval, while the last one is analyzed in an IPG pH 3.5-5 span. The patterns are specific for distinct populations, inter- and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed-bed, carrier-ampholyte (CA)-IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein-IPG matrix interaction (Electrophoresis, 1987, 8, 62-70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.     

Microchip with an open tubular immobilized ph gradient for UV whole column imaging detection

This study reports a new method for establishing an open tubular IPG in a microchip coupled with a whole column image detection (WCID) system for protein separation applications. This method allows a wider range of immobilized pH (2.6–9.5) to be established in a PDMS/quartz channel by controlling the diffusion of acidic and basic polymer solutions into the channel through well‐designed channel dimensions. The developed pH gradient was experimentally validated by performing the separation of a mixture of standard pI markers. It was further validated by the separation of the hemoglobin control AFSC sample. This method is advantageous over existing IPG methods because it has a wider range of pH and maintains the open tubular feature that matches the UV WCID to improve the sensitivity.     

Detection of the hypusine-containing protein (HP = eIF-5A) in crude yeast extracts by two-dimensional western blots.

The hypusine-containing protein (HP) with its unique modification of a specific lysine residue resulting in the amino acid hypusine is highly conserved among all eukaryotes and is also found in Archaebacteria. Studies of the protein function in translational processes showed a stimulatory effect in the methionyl puromycin assay, but not in in vitro translation of native mRNA. It was therefore also designated as eIF-5A. To further investigate the role of HP in cellular metabolism, we purified the protein from Saccharomyces cerevisiae and raised polyclonal antibodies in chicken. Immunoglobulin preparations from the eggs of the immunized hens were used for Western blot analysis of HP in crude yeast extracts. For those studies, the soluble protein fraction of the yeast was resolved on two-dimensional gels (first dimension: isoelectric focusing using an immobilized pH gradient (IPG), pH 4-7; second dimension: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 12% T) and subsequently blotted onto Fluorotrans membrane. Anodic versus cathodic application of the extracts of the IPG strips was compared.     

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.     

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.     

pI-based phosphopeptide enrichment combined with nanoESI-MS

IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pI values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to pI is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Ultrasensitive electrochemical immunoassay based on counting single magnetic nanobead by a combination of nanobead amplification and enzyme amplification

An ultrasensitive electrochemical immunoassay method based on counting single magnetic nanobead (MNB) with combined amplification of nanobead and enzyme. Carcinoembryonic antigen (CEA) and MNB were initially immobilized on substrate at 1:1 molar ratio through sandwich immunoreactions. The MNBs were then labeled with alkaline phosphatase (AP) and continuously introduced to capillary with disodium phenyl phosphate (DPP). APs convert DPPs into a phenol zone around each moving MNB, i.e., one CEA. The phenol zones can be electrochemically detected as peaks and counted for CEA quantification. The detection limit for CEA is 5.0 × 10^?17 mol/L.