Separation of acidic proteins by capillary zone electrophoresis and size-exclusion high-performance liquid chromatography: a comparison (original) (raw)
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Journal of Chromatography A, 1997
The use of phytic acid to improve protein analysis by capillary electrophoresis (CE) is becoming more and more popular, Due to its size and number of negative charges (up to 12) it provides a high ionic strength combined with a low conductance resulting in an efficient decrease of wall adsorption for proteins. Because of its twelve acidic groups, phytic acid can be used as a buffer over a wide pH range (pH 2-11). The limited wall adsorption of proteins using phytic acid-containing buffers is observed for buffers with a pH of 5.5 and higher. With a monoprotic buffer, most of the investigated proteins show wall adsorption at the pH values studied. In case of a phytic acid buffer, wall adsorption is reduced by a factor of 2-4. The use of phytic acid both as a modifier and as a pH buffer results in more pronounced differences between the various protein mobilities compared with the use of monoprotic buffers. As a result this feature can be used to improve resolution in protein separations.
Journal of Chromatography A, 1994
A neutral, hydrophilic coated capillary with negligible electroosmotic fow was characterized as to migration time reproducibility and separation cffkiency for protein separation by capillary zone electrophoresis. Consecutive runs (over 200 runs) of the basic proteins at pH 6.0 yielded excellent migration time reproducibility (< 2% R.S.D.) and high separation efficiency (ca. 3-S. 10' plates/m). The acidic proteins were separated at pH 8.0 under the reversed polarity (cathode at the injection end), and excellent migration time reproducibility of less than 3% was achieved. Separation of egg white proteins at pH 3.0 showed migration time reproducibility of less than 0.5% R.S.D. (n = 36) for lysozymc, conalbumin and ovalbumin.
Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis
ELECTROPHORESIS, 2004
Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.
Selectivity in capillary electrophoresis: the use of proteins
Journal of Chromatography A, 1997
Proteins, by their very diverse nature, provide a wide variety of options for generating selectivity in capillary electrophoresis (CE). Their use in different modes of CE will be considered in this review. Proteins added in solution to the background electrolyte allow separations to be made in a similar fashion to other electrokinetic chromatography methods, e.g., micellar separations. Alternatively, different immobilization schemes can be used to secure proteins within the capillary; these have included capillary electrochromatography with the protein grafted onto a silica support, or immobilization of the protein within a gel structure. Compounds varying in size from small inorganic ions to biopolymers may be bound by proteins. There is the potential for any sort of intermolecular interaction to play a role in the binding process (e.g., hydrophobic interactions, electrostatic interactions, etc.). Very specific high-affinity binding often occurs, but also there is often weaker, non-selective binding. Frequently the interactions of chiral compounds with proteins are stereoselective. Obtaining chiral selectivity has been one of the main applications of protein selectors in CE, and this use will be emphasized here in a discussion structured by type of protein. As well as utilizing the selectivity of proteins to develop separations, the role of CE in investigating ligand-protein interactions will be emphasized.
ELECTROPHORESIS, 2004
However, in a different study it was concluded that CE and RP-HPLC can give equivalent results and can be used for cross-validation studies [16]. RP-HPLC has also been successfully employed in the monitoring of the stability of insulin. Insulin can form two main degradation products, i.e. the monodesamido-A21-insulin and the monodesamido-B3-insulin that are formed under acidic and neutral conditions, respectively. In Fig. 1 an example of such a separation is shown. In this example a C-18 column and a mobile phase of 0.2 M sodium sulphate buffer (pH 2.3) with acetonitrile was used. Affinity chromatography Affinity chromatography is sometimes referred to as biospecific interaction chromatography. This is a technique that is based on specific interactions between molecular pairs from biological systems. Examples of ligands that can be used are antigens, antibodies, hormones, cofactors, receptors etc. This means that it is possible to tailor make a column with affinity only for the analyte of interest, i.e. only this is retained on the column and the rest is eluted. At the end an elution buffer, which disrupts the ligand/protein bond, is applied to rinse the analyte from the column and (ideally) obtain a pure product. This can be very cost effective since as much as 50-80% of the total direct production cost of manufacturing a therapeutic product comes from the final steps, i.e. purification and polishing of the product [29]. Therapeutic proteins often contain mixtures of isoforms, which originate from post-translational modifications in the host cell expression system, e.g., glycosylation, sulphation, oxidation etc. [29]. Since some ligands employed in affinity chromatography originate from natural sources themselves they must also be purified, since they may contain host DNA, and viruses and often show lot-to-lot variation. One way to reduce these problems is to use synthetic ligands such as the Active Red K2BP that were immobilized as affinity ligands on a chemically cross-linked cellulose film. Using this membrane they managed to purify commercially available calf intestinal alkaline phosphatase about 40 times and the recovery was approximately 60%. The purification of calf intestinal alkaline phosphatase and human urine urokinase by purpose-designed affinity ligands attached to Dynospheres XP-3507 was described by Clonis and Lowe [33]. They also compared the purpose-designed ligands with two other reference ligands, i.e. 6-aminohexyl C.I. Reactive Blue 2 and 4-aminobenzyl phosphonic acid that are known to interact with alkaline phosphatase and urokinase, respectively. Their conclusion was that the purpose-designed ligands were much better than the reference ligands in purification and recovery. Roy and Gupta have described the purification of alkaline phosphatase from chicken intestine by affinity chromatography [34]. The purification progress was followed by enzymatic activity assays where a substrate (para-nitrophenyl phosphate) was converted to a coloured product (paranitrophenol) that was measured spectrophotometrically. In Fig. 4 a reproduction of results presented by the authors is given. 1.2.3 Capillary electrophoresis Electrophoresis methodologies are the most commonly used in the separation of proteins, and are the methods of choice for the majority of protein chemists. However, for large-scale procedures (milligram and gram quantities) liquid chromatographic techniques are commonly preferred, since these have larger loading capacity. For purification of small amounts of polypeptides polyacrylamide gel electrophoresis (PAGE) offers the best results. Electrophoretic methods have unsurpassed resolving power and speed and are suitable for the analysis of both hydrophobic as well as hydrophilic peptides and proteins. Capillary electrophoresis (CE)
Analytical Chemistry, 2002
To improve the separation efficiency while achieving high sensitivity for the analysis of proteins' microheterogeneity, a segmental-filling technique has been developed and tested in capillary electrophoresis with laser-induced native fluorescence using a pulsed Nd:YAG laser. Using a short plug of SDS applied to the capillary and the anticonvectant poly(ethylene oxide) (PEO), the microheterogeneities of a number of proteins with pI values ranging from 4.5 to 11.1 could be detected. This high resolving power is due to reduced adsorption on the capillary wall, sieving, and the interaction with SDS. Consequently, the length and the concentration of the SDS plug play a significant role in determining the resolution and sensitivity. The method has been applied to the analysis of salivary and cerebrospinal fluid (CSF) samples. Without any sample pretreatment, using a 10-s 1× SDS plug, six r-amylase isoforms in a salivary sample were resolved in 17 min and three more peaks were detected in a CSF sample. With simplicity, high resolving power, and rapidity, the method has shown great potential for proteomics.
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.
Journal of Chromatography A, 1995
The electrophoretic mobilities of bovine serum albumin,/3-1actoglobulin A and B, a-lactalbumin and myoglobin were measured in free solution using an improved version of the Boltz-Todd vertical density-gradient electrophoresis column. Dialysis membranes were used for the isolation of the side-arm electrodes from the column and large-volume electrode containers were connected to each other by a circulating buffer loop. The improvements increased reliability, facilitated removal of electrode gas, prevented proteins from contacting electrodes and allowed the use of low conductivity buffers without ion depletion. A low conductivity buffer (Tris-glycine) allows the use of high fields for rapid separations. The apparatus is modular and allows easy modification of column dimensions. We have also measured the electrophoretic mobility of these proteins in a coated capillary in the absence of significant electroosmotic flow.