Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. III: pH 2.5-4 intervals

To study the molecular mass distribution and number of species in narrow-range (2-pH-unit wide, in the nominal pI 2-4 or 3-5 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted, consisting of a preparative focusing step in a Rotofor instrument, followed by analysis of every other collected fraction (10 out of 20) by CE-MS. It was found that Ampholine pH 3.5-5 contains 105 different molecular mass (M(r)) compounds, in the M(r) interval 205-965 Da, for a total of 446 isoforms. Bio-Lyte pH 3-5 consists of 84 different M(r) species, in the M(r) range 216-965 Da, for a total of 383 isoforms. Servalyt pH 2-4 is made of 227 different M(r) compounds, in the M(r) interval 204-929 Da, for a total of 1201 isoforms. Pharmalyte pH 2.5-5 comprises 245 amphoteres, in the M(r) range 203-857 Da, for a total of 857 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and almost no 'poor' species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value. Due to some overlap with the adjacent acidic pH 4-6 interval, the species in common have been evaluated: the most extended overlaps are found in Ampholine (55% of the species appearing in the two neighbouring intervals) and in Servalyt (47% coincidence). The lowest overlaps are found in Pharmalyte (23%) and in Bio-Lyte (20%).     

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. IV: pH 6-8 intervals

Four commercial brands of carrier ampholytes (Ampholine, Pharmalyte, Servalyt, Bio-Lyte), in the pH 6-8 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by CE-MS of 10 out of 20 fractions harvested, in the second dimension. The findings: Ampholine (pH 6-8) contains 80 different M(r) compounds, in the M(r) interval 216-979 Da, for a total of 326 isoforms. Bio-Lyte (pH 6-8) consists of 62 different M(r) species, in the M(r) range 341-1048 Da, for a total of 237 isoforms. Servalyt (pH 6-8) is made of 126 different M(r) compounds, in the M(r) interval 240-785 Da, for a total of 703 isoforms. Pharmalyte (pH 5-8) comprises 123 amphoteres, in the M(r) range of 221-992 Da, for a total of 476 isoforms. Pharmalyte appears to be the best brand, with a good proportion of species focusing sharply at their pI position and relatively few 'poor' species, distributed along the entire pH gradient. General conclusions are drawn on the properties of all the pH intervals explored in this series of investigations and some guidelines for possible synthetic routes ameliorating the neutral and alkaline pH intervals are discussed.     

Carrier ampholytes for IEF, on their fortieth anniversary (1967-2007), brought to trial in court: the verdict

The present review summarizes the data accumulated in 1-year work, by exploring, via a 3-D methodology (Rotofor fractionation followed by CE-MS), all the narrow (2 pH unit wide) carrier ampholyte (CA) compounds for IEF, produced by three companies (Pharmacia with Pharmalyte and Ampholine, BioRad with Bio-Lyte and Serva with Servalyt). All species have been assessed by measuring the types of pH gradient produced, the total number of individual chemicals (with M(r) values) and isoforms and their focusing behavior ('good' or 'poor' ampholytes). Servalyt contains a grand total of 686 chemical entities and no less than 3899 isoforms; Pharmalyte 643 and 2211; Bio-Lyte 255 and 1192; Ampholine 294 and 1182, respectively. In terms of M(r) distribution, although all 2-pH-unit ranges start with the same low M(r) values (ca. 200) their upper limits are quite different. Thus, Pharmalyte reaches an upper M(r) value of 1179 (in the pH 4-6 range), versus 907 for Servalyt, 835 for Bio-Lyte and 893 for Ampholine. In general, in going towards the more alkaline pH intervals (e.g. pH 8-10) the molecular mass of carrier ampholytes (CAs) is reduced to as low as 491 (Bio-Lyte), indicating that the alkaline species are probably made with shorter oligoamines and are, in general, less substituted. All acidic pH intervals (up to pH 6-8) appear to be constituted by a very large proportion of well focusing species, indicating small values of DeltapK across their pI. Above pH 8, all brands of CAs worsen, the vast majority being unable to focus properly and sustain adequately the pH gradient. General guidelines are given for the synthesis of new alkaline species for improving the basic pH ranges.     

Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results

In an attempt to prepare quasi-isoelectric buffers as BGEs for CE, carrier ampholytes (CAs) (Ampholine, pH 7-9; Servalyt, pH 7-9; Bio-Lyte, pH 8-10 and Pharmalyte, pH 8-10.5) have been subdivided with the Rotofor into 20 fractions, of ca. 0.1 pH unit span, whose composition has been studied by CZE-MS. The results have allowed identifying the number of different molecular mass compounds present in every commercial brand, as well as the number of isoforms (having identical mass, but representing positional isomers) associated with a given M(r) value. Ampholine is composed of 29 species, for a total of 85 different isoforms; Bio-Lyte is made of 43 compounds, for a total of 136 isoforms; Pharmalyte comprises 58 different M(r) chemicals, for a total of 102 isoforms and Servalyt is constituted by 65 species, for a total of 306 compounds (all of these values to be considered as minimum numbers, as detected by the present methodology). Surprisingly, and contrary to theory, a very large proportion (up to 70%) of these species are 'poor carrier ampholytes', in that they are unable to focus and are evenly distributed along the generated pH gradient in the electric field. Paradoxically, the pH gradient is created and sustained by the minority of species (30% for three brands, up to 50% for Pharmalyte) that appear to focus at their pI position into reasonably sharp zones. Even in the narrowest pI fraction, up to 20 different compounds can be detected. It is concluded that very few amines with different useful pK values are utilized for the synthesis and that a new generation of CAs with a more diversified population of amines with proper pK values within the given pH intervals should be sought. Ampholine, the poorest of the commercial brands, appears to be still made with the original synthesis devised by Vesterberg, i.e. by reacting a concoction of oligoamines with alpha,beta-unsaturated acids.     

Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9-11 interval

As a last part of an investigation on all 2-pH-unit intervals of carrier ampholytes (CAs) for IEF (see Electrophoresis 2006, 27, 3919-3934; 2006, 27, 4849-4858; 2007, 28, 715-723) two different lots of Servalyt CAs, in the pH 9-11 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by capillary electrophoresis mass-spectrometry of 10 out of 20 fractions harvested, in the second dimension. The findings: the two lots contain 65 and 69 different M(r) compounds, in the M(r) interval of 232-667 Da, for a total of 341-387 isoforms, respectively. Since this is a chaotic organic synthesis, the high reproducibility (here demonstrated for the first time during the 40 years of existence of CAs) of the synthetic process (for two batches produced at 6 years of distance) is remarkable, considering that a 94% agreement for the individual chemicals and 88% agreement for the total number of isoforms for the two lots is found. It is additionally demonstrated that the lower pI species are accompanied by considerably more isoforms than the high pI forms and that in all cases such isoforms consist of family of compounds clustered around the pI of the parental form, with a pI spread of ca. 0.1-0.2 pH units.     

Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing

An isoelectric point (pI) calibration kit containing fish muscle parvalbumins was prepared and tested for its suitability for isoelectric focusing (IEF) in the presence of 8 M urea. The pattern obtained by urea CleanGel IEF consisted of nine bands covering the pI range 4.96-5.64. This range is relevant for species identification of heated fish by urea IEF. The kit may also be used for native IEF in the low pH range, as demonstrated by running an extract made from the kit together with water-soluble fish muscle proteins on Servalyt Precotes 3-6.     

Different patterns of human serum transferrin on isoelectric focusing using synthetic carrier ampholytes or immobilized pH gradients

Isoelectric focusing (IEF) of human serum transferrin allows splitting of the protein pattern into three forms corresponding to the diferric, monoferric and apoform. A detailed analysis of this pattern, performed on transferrin at different degrees of iron saturation, demonstrated that free Ampholine carrier ampholytes (CA) alter the expected results, always giving a complex pattern with multiple bands. In particular, the monoferric form appears to be the predominant one, regardless of the starting saturation of transferrin. In contrast to IEF-CA, the new technique of IEF in immobilized pH gradients (IPG), shows a much simpler pattern with the same samples. Moreover, the different transferrin forms are focused at the same pI values as in IEF-CA but the pattern appears to correspond to the expected distribution. IPG analysis gives a pattern similar to IEF-CA when free Ampholine CA are added either to the samples and/or as electrode solutions. A chelating action of Ampholine CA on Fe+3 might be responsible for these effects, while Immobilines, due to their different chemical nature or to the different focusing procedure, are not able to interact with iron.     

Microheterogeneity of apolipoprotein D as revealed by electroblotting following isoelectric focusing in Immobiline DryPlates.

The microheterogeneity of apolipoprotein D was examined by a procedure involving, in sequences: (i) electrophoresis in an immobilized pH 4-7 gradient in an Immobiline DryPlate-polyacrylamide gel supplemented with Ampholine pH 5-7, (ii) covering of the gel with sodium dodecyl sulfate-containing agarose, (iii) electroblotting onto a polyvinylidene difluoride membrane and (iv) immunological identification. Seven isoforms were obtained with partially purified apolipoprotein D. Using this technique the apparent pI values at 15 degrees C for the isoforms were 4.57, 4.67, 4.78, 4.83 and 5.95, 6.06 and 6.19 (SD +/- 0.05 for all). Direct staining of the Immobiline DryPlate could not reveal the isoforms of partially purified apolipoprotein D.     

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.     

Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric membranes.

A purified, size-homogeneous (100 kDa), desialylated form of a truncated, soluble form of the epidermal growth factor receptor secreted by A431 human tumor cells has been found, by isoelectric focusing in immobilized pH gradients, to consist of two major isoforms (with pIs of 6.96 and 6.71), one intermediate form (pI 6.45) and a number (> 10) of minor components. The two major components have been purified to charge homogeneity by isoelectric focusing in a multicompartment electrolyzer with buffering isoelectric membranes having the following pI values: 5.90, 6.63, 6.76, 6.92, 7.05 and 7.35. Such single pI species are presently used for attempts at crystal growing.     

Repeatedly usable immobilized pH gradient in a monolithic capillary column

Glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were used to synthesize a monolithic capillary column containing reactive epoxy groups. Glutaraldehyde was introduced and linked to the monolith after a process of amination. An aqueous solution of commercial carrier ampholytes (CAs, Ampholine) was focused in such a polymer column. The primary amino groups of CAs reacted with glutaraldehyde along the capillary. CAs were immobilized at different positions in the column according to their isoelectric points (pI), resulting in a monolithic immobilized pH gradient (M-IPG). Isoelectric focusing (IEF) was performed without CAs in such an M-IPG column. Due to the covalent attachment of the CAs this M-IPG can be repeatedly used after its preparation. Good stability, linearity, and reproducibility were obtained.     

New azo dyes as colored isoelectric point markers for isoelectric focusing in acidic pH region

Newly prepared azo compounds and several commercially available indicators were investigated for their applicability as colored isoelectric point (pI) markers for isoelectric focusing (IEF) in the acidic range below pH 5. The majority of compounds described here can serve as primary standards since their pI values were determined by UV-VIS spectrophotometry independently IEF and direct measurement with a pH electrode. Subjected to gel IEF they show narrow and well-observable zones of different colors. Finally, our work resulted in suggestion of a color ladder composed of pI markers covering the pH range from 1.5 to 4.7.     

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

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.     

Protein adsorption in fused-silica and polyacrylamide-coated capillaries

The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.     

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.     

Studies on an apolipoprotein C-II variant occurring in Caucasians.

Apolipoproteins C (apo C-II, apo C-III0, apo C-III1 and apo C-III2) from delipidated very low density lipoproteins (VLDL) of 522 normo- and hyperlipoproteinemic Caucasians were screened by analytical isoelectric focusing. The immobilized pH gradient used was pH 4.0-5.0 with 7 M urea, which raised the apparent pH range to 4.8-5.7. As identified by immunoblotting, six unrelated persons had two major isoforms of apo C-II, the normal apo C-II-1 (which focuses between apo C-III0 and apo C-III1) and a variant, designated apo C-II-v according to Huff et al., focusing between apo C-III1 and apo C-III2 due to a more acidic pI. In narrow pH gradients, apo C-II-v can readily be discriminated from the minor isoform, apo C-II-2, due to its slightly more basic pI, corresponding to a difference of 0.01 pH units. Neuraminidase treatment did not alter the pI of apo C-II-v and on two-dimensional electrophoresis the molecular weights of apo C-II-1 and apo C-II-v were indistinguishable. The frequency of apo C-II-v was 1.2%. It was the same in males and females and was independent of hypertriglyceridemia. The autosomal codominant inheritance could be demonstrated in the pedigree of one family. Electroblotting of apo C-II-1 and apo C-II-v onto activated glass fiber sheets, followed by amino acid sequence analysis of the amino terminal ends, revealed an exchange of the amino acid lysine at position 19 by threonine in apo C-II-v.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolving isoforms of aldose reductase by preparative isoelectric focusing in the Rotofor

We have resolved and characterized isoforms of aldose reductase from bovine and porcine lenses by preparative isoelectric focusing with narrow pH gradients using the Rotofor. Both bovine and porcine lens aldose reductases were resolved as two enzyme isoforms. The bovine isoforms were Mr40400 +/- 445 polypeptides of pI4.71 and 5.19. Porcine isoforms were Mr41500 +/- 450 polypeptides of pI 4.90 and 5.30. Staphylococcus aureus V-8 protease digestion patterns for each set of isoforms were essentially identical and all isoforms probably contain blocked amino terminal amino acids. Antiserum to bovine lens aldose reductase cross-reacted with porcine lens aldose reductase. Each isoform displayed substrate preferences characteristic of mammalian aldose reductases. With purification, both bovine and porcine lens aldose reductases became less sensitive to inhibition by 6-fluoro-spiro-(chroman-4.4'-imidazolidine)-2',5'-dione (sorbinil).     

Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants†

One pressure and three chemical mobilization strategies have been optimized and tested for two‐step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1–10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3–10 with Pharmalyte 5–6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5–6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l ‐Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l ‐Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.