Isoelectric focusing of proteins and peptides in gel slabs and in capillaries (original) (raw)
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Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid
Electrophoresis, 1997
Capillary zone electrophoresis in stationary, isoelectric buffers is a novel method for generating peptide maps of protein digests. The buffer system developed is composed of iminodiacetic acid (IDA), whose physico-chemical parameters were foundby theoretically modeling and experimental verificationto be: pf 2.23 (at 100 mM concentration), pK, = 1.73 and pK, = 2.73 (no attempts were made at measuring the pK of the primary amino group, since such a low pf value would be compatible with any pK value of the basic group, down to as low as pK 5.5). IDA is compatible with most hydro-organic solvents, including trifluoroethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 mM IDA, 10% TFE and 0.5 O/ o hydroxyethylcellulose (HEC) allows generation of peptide maps with high resolution, reduced transil limes and no inleraction of even large peptides with the wall. However, the best background electrolyte was found to be a solution of 50 mM IDA in 0.5% HEC and 6-8 M urea, one of the best solubilizers of proteins and peptides known. In this last electrolyte system, peptide maps of 0-casein digests (known to contain also very large peptides, up to 6000 Da) could be generated with excellent resolution and half the transit times as compared with the standard buffer adopted in peptide analysis (80 mM phosphate buffer, pH 2.0). IDA thus appears to be another valid isoelectric buffer system, operating in a different pH vvindow (pH 2.33 in 50 mM IDA) as compared to the other amphotere previously adopted (50 mM Asp, pH 2.77) for the same kind of analysis.
The present review offers a survey of modern isoelectric focusing (IEF) techniques, including the conventional one in soluble amphoteric buffers (CA-IEF) and immobilized pH gradients (IPG). In the case of CA-IEF, recent advances on its use as a ®rst dimension for two-dimensional electrophoresis are covered. The properties of isoelectric buffers, in terms of buffering power and conductivity, are reviewed in view of their importance as background electrolytes in capillary zone electrophoresis (CZE). In the case of IPGs, examples are given of the high correlation between theoretically predicted and experimentally measured pI values. New aspects of IPGs, such as focusing in extremely alkaline pH intervals, and its interfacing with mass spectrometry, are described. In the case of capillary IEF, new aspects of the technique are described, in particular: (a) how to modulate the slope of the pH gradient for increasing resolution; and (b) how to keep proteins in solution at, and in the proximity of, the pI value. The review ends with an excursus on the use of isoelectric buffers in capillary zone electrophoretic separations. Such buffers offer unique advantages: they permit very high voltage gradients (up to 1000 V/cm) and, thus, minimize analysis times (down to a few minutes in 30±35 cm long capillaries). This results in a marked increment in resolution due to minimal diffusion-driven peak spreading. Such buffers are ®nding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of intact proteins. #
Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2006
Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.
1999
High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 pm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with a-a-diamines such as ethylenediamine or 1,lO-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400 000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to l 900 000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.
Divergent-flow isoelectric focusing for separation and preparative analysis of peptides
ELECTROPHORESIS, 2012
A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.
Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation
Analytical Chemistry, 2007
Capillary zone electrophoresis and carrier ampholytes based capillary electrophoresis have been used as a second separation step to Off-Gel isoelectric focusing for the analysis of complex peptide mixtures. A tryptic digest of four proteins (bovine serum albumin, -lactoglobulin, horse myoglobin, cytochrome c) has been chosen as a peptide test mixture. After assessment of different modes of capillary electrophoresis as a second dimension to Off-Gel isoelectric focusing, the optimized two-dimensional platforms provide a degree of orthogonality comparable to state-of-the-art multidimensional liquid chromatography systems as well as a practical peak capacity above 700.
Electrophoresis, 2004
The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5–10 μM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.
Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis
ELECTROPHORESIS, 2008
The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. a-Lactalbumin (14 kDa, pI 4.8) and a-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.