Determination of the isoelectric point of proteins by capillary isoelectric focusing

Different ways of determining isoelectric points (pI) of proteins in capillary isoelectric focusing are reviewed here. Due to the impossibility of direct pH measurements in the liquid phase, such assessments have to rely on the use of pI markers. Different types of pI markers have been described: dyes, fluorescently labelled peptides, sets of proteins of known pI values. It appears that, perhaps, the best system is a set of 16 synthetic peptides, trimers to hexamers, made to contain each a Trp residue for easy detection at 280 nm. By a careful blend of acidic (Asp, Glu), mildly basic, with pK around neutrality (His), and basic (Lys, Arg) amino acids, it is possible to obtain a series of pI markers with pI values quite evenly distributed along the pH scale, possessing good buffering capacity and conductivity around their pI values and thus focusing as sharp peaks. Another approach to pI determination is the monitoring of the current during mobilization: this allows, with the aid of known pI markers, to calibrate the system with a pI/current graph. Pitfalls and common errors in pI determinations are reviewed here and guidelines given for minimizing such errors in pI estimation.     

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Sixteen peptides (trimers to hexamers) were designed for use as a set of pI markers for capillary isoelectric focusing (CIEF). Each peptide contains one tryptophan residue for detection by UV absorption and other amino acid residues having ionic side chains, which are responsible for focusing to its pI. The pIs of these peptides were determined by slab-gel IEF using commercial carrier ampholytes. The focused peptides in the gel were detected by absorption measurement at 280 nm using a scanning densitometer and the pH gradient was determined by measuring the pH of the gel using an oxidized metal membrane electrode. The pI values of the peptides ranged from 3.38 to 10.17. The obtained values agreed well with the predicted ones, which were calculated based on amino acid compositions, with root mean square differences of 0.15 pH unit. The peptides were detected at 280 nm as very sharp peaks when separated by CIEF. The pI values of some standard proteins were redetermined by CIEF by using this set of peptide pI markers and the values agreed closely with those reported previously. The sharp focusing, stability, high purity and high solubility of these synthetic pI markers should facilitate the profiling of a pH gradient in a capillary and the determination of the pI values of proteins.     

固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

通过聚合物原位聚合反应，制备了部分填充的毛细管整体柱。pH 3~10的载体两性电解质被固化在该毛细管整体柱上。在引入八通进样阀、三通阀和四通连接单元的基础上，构建了适用于固化pH梯度毛细管等电聚焦整体柱（M-IPG）的平台。在蛋白质药物测定过程中，用M-IPG柱和羟丙基纤维素（HPC）涂层毛细管柱同时对曲托珠单抗和依那西谱的等电点进行了测定。结果表明，两种等电聚焦柱都能够同时分离混合蛋白质样品并测定蛋白质类药物中单抗和融合蛋白质的等电点（pI），M-IPG柱所测的pI值与HPC涂层毛细管柱测定的结果基本一致，表明了该柱在进一步构建多维分离平台进行蛋白质组学研究方面的潜力。     

Peak identification in capillary isoelectric focusing using the concept of relative peak position as determined by two isoelectric point markers

In CIEF analysis, sample peaks can be identified by their relative peak positions (RPP) that are determined using only two internal pI markers. The two internal pI marker peaks should bracket, as close as possible, the sample peaks. The RPP values of the sample peaks are then calculated using the pI values, peak positions of the two pI markers, and peak position of the sample. Use of this method can effectively compensate for pH gradient distortions that often occur as a result of salts. Also, as shown by the results of this paper, regardless of the linearity of the pH gradient established by the given carrier ampholytes, sample peaks can be identified within an SD of 0.1 pH unit in RPP (<2% RSD) as long as the sample is run using the same carrier ampholytes and maintaining salt concentrations in the range of 0-15 mM.     

A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing

Human carbonic anhydrase (hCA) IX and XII are isoenzymes which are highly overexpressed in many cancer types. Recently, it has been shown that hCA IX contributes to the acidification of the tumor environment leading to chemoresistance with basic antitumoral drugs. The development of selective hCA inhibitors constitutes a new therapeutic axis. In order to elucidate the specific interactions between hCA and inhibitors, physico-chemical properties of hCA must be evaluated. This work reports the determination of the isoelectric point (pI) of a series of hCA isoforms by capillary isoelectric focusing. First, the method was optimized with synthetic UV-detectable pI markers using a central composite design. The separation was performed in a fused-silica capillary chemically derivatized with hydroxypropylcellulose and using a glycerol-water medium as the anticonvective gel. Three main factors (ampholyte content, focusing time and mobilization pressure) were optimized in order to obtain the best resolution, detection threshold and precision on the pI determination. Then, the model was validated through the analysis of standard proteins mixture having known pI values, before investigating the pI of hCA isoforms.     

毛细管等电聚焦和电渗泵驱动聚焦区带分离蛋白质

建立了一种利用电渗泵驱动毛细管内的聚焦区带,实现毛细管电泳等电聚焦分离蛋白质的方法。通过控制电压来调节泵的输出流量,从而调节聚焦区带的迁移速度。适用于毛细管电泳等电聚焦两步法分离蛋白质等两性物质。考察了对牛血清白蛋白和溶菌酶两种粗提蛋白质混合物的分离,迁移时间的RSD分别为1.6%和1.3%,峰面积的RSD均为1.6%,证明方法可行。     

Fluorescein-based pI markers for capillary isoelectric focusing with laser-induced fluorescence detection

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.     

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.     

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing.

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol-H2O2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethyl-cellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6 x 10(-9) M and the linear dynamic range was greater than two-orders of magnitude (up to 2 x 10(-6) M).     

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.     

Recent applications of capillary isoelectric focusing

After the advent of capillary isoelectric focusing (CIEF) in the 80's several approaches have been developed in order to use the technique in routine analyses. The recent years showed an extensive increase in the applications of this technique employing its exceptionally high-resolution power. Methodological improvements, as well as hyphenation with other electrophoretic and chromatographic separation procedures, proved the versatility of CIEF in studies of clinically important proteins, recombinant product, cell lysates and other complex mixtures. The combination of CIEF with mass spectrometry detection is one of the major challenges for studying proteomics. This review collected the recent applications of CIEF including innovations in the experimental setup, remedies for the presence of salts in samples, calibration of the pH gradient, carrier ampholyte-free isoelectric focusing, the progress in micropreparation, two-dimensional separations, etc.     

Miniaturization of capillary isoelectric focusing.

Miniaturization of whole-column imaging capillary isoelectric focusing (CIEF) is discussed. A 1.2 cm capillary was used as a separation column for CIEF. The experimental results for the analysis of two pI markers and the protein myoglobin showed that good CIEF separation results could be obtained. Secondly, a light-emitting diode (LED) was used as the light source for the whole-column absorbance imaging detection. The focusing of both the pI markers and myoglobin were observed with the LED light source. The whole-column imaging CIEF instrument was simplified and miniaturized by the use of the LED. Further developments are also discussed.     

Application of capillary isoelectric focusing and peptide mass fingerprinting in carbohydrate-deficient transferrin detection

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse and is widely used in clinical diagnosis to detect and follow up excessive alcohol consumption. However, false %CDT results still exist in CDT detection, because of interference from genetic variants and the lack of standardization in CDT analysis. Therefore, it is still very important to find a method with high sensitivity and high accuracy for CDT detection. Here, we compared the detection sensitivity and accuracy of pI values based methods [isoelectric focusing on polyacrylamide gel electrophoresis (IEF-PAGE) and capillary isoelectric focusing (CIEF)] with hydrophobic characteristic based methods [reversed-phase high-performance liquid chromatography (RP-HPLC)] on CDT detection. Moreover, we investigated the potential of peptide mass fingerprinting (PMF), a method based on the mass spectrometry to identify human transferrin (HTf) variants including CDT isoforms and genetic variants, based on their specific peptide masses. Results indicated that PMF can identify HTf variants including CDT isoforms and genetic variants based on their specific peptides, and CIEF showed higher sensitivity detection of HTf variants than RP-HPLC and IEF-PAGE did. Accordingly, we suggest that PMF is suitable for identifying CDT with high accuracy, and CIEF has potential for detection of CDT and genetic variants with high sensitivity; moreover, they are both worth further investigation in clinical diagnosis.     

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.     

Separation of human cerebrospinal fluid proteins by capillary isoelectric focusing in the absence of denaturing agents

Conditions of capillary isoelectric focusing (CIEF) to separate human cerebrospinal fluid (CSF) proteins were examined referring to those which we have established for the separation of human plasma/serum proteins. Since the average protein concentration in CSF is about 1/200 of plasma and the salt concentration is at almost the same level as plasma, desalting of CSF samples with minimum dilution was a prerequisite for CIEF analysis of CSF proteins. We constructed an apparatus to dialyze CSF at the level of 20-30 microL, this volume being sufficient for 3-4 repeated analyses of the CSF sample. To trace the process of dialysis, a simple device to measure the conductivity of the dialyzate was also constructed. Most of the CIEF conditions for plasma protein analysis could be applied for CSF protein analysis. However, the addition of N,N,N',N'-tetramethylethylenediamine (TEMED) at a suitable concentration was necessary to improve the resolution of basic proteins (IgG region), since some CSF patterns showed peaks of basic proteins which are not obvious in the serum of the same patient. About 70 peaks and shoulders of CSF proteins could be detected by the established CIEF technique. The results of CIEF analysis of CSF samples suggested that the technique will be useful as a survey method to detect specific proteins in CSF, which might relate to disorders in the central nervous system.     

Determination of isoelectric points of acidic and basic proteins by capillary electrophoresis

The isoelectric point (pI) of a protein is of practical importance in many separation procedures, both analytical and preparative. The pI is defined as the pH where the net charge of the protein is zero. Therefore, by plotting the mobilities of the proteins against pH, the intercept at zero mobility should yield the pI value. Isoelectric points have traditionally been determined by isoelectric focusing. In this paper, the potential of capillary electrophoresis as an alternative technique for the determination of pI values of both acidic and basic proteins was investigated. The problem commonly encountered with adsorption of the positively charged proteins with the unprotonated silanol groups of the fused-silica wall is solved by applying a dynamic coating of a polycationic reagent to the wall. The advantages of this technique of determining the pI values are simplicity, speed and minimal sample requirement.     

Isoforms analysis of recombinant human erythropoietin by polarity-reversed capillary isoelectric focusing

Recombinant human erythropoietin (rhuEPO) has been extensively used as a pharmaceutical product for treating anemia in the clinic. Glycosylation of rhuEPO was crucial for affecting biological activity, immunogenicity, and pharmacokinetics. Because of the heterogeneity of glycan, the structure of rhuEPO was complex with several isoforms. Characterization of isoforms was important for quality control of rhuEPO. Here, an improved cIEF method has been established and validated. A polarity-reversed focusing step was used by reversing both the polarity of the voltage and the catholyte and anolyte vials. A weak base (100 mM ammonium hydroxide solution) was used as a chemical mobilizer to make the acidic bands mobilize stably to the detection window. Compared with CZE method in European Pharmacopoeia, the numbers of isoforms and their peak area percentage were highly consistent. Better reproducibility and higher resolution have been obtained by the improved cIEF method. Moreover, in improved cIEF method, the isoelectric points (pI) of each isoform can be calculated and used for identification. It was also the first time that the cIEF method was fully validated for rhuEPO analysis according to the International Conference on Harmonization (ICH) guidelines.     

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78–4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.     

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Estimation of biotinylated lectin by isoelectric focusing

Concanavalin A (Con A) was biotinylated to various degrees using N-biotinyl-omega-aminocaproic-acid-N-hydroxy succinimide ester as the biotinylation reagent, and then analyzed by isoelectric focusing using PhastGel IEF 3-9. The isoelectric points of biotinylated ConAs were found to decrease with increasing concentration of the biotinylation reagent. Analysis by isoelectric focusing followed by dot blotting clearly indicated that the biotinylated ConA with an isoelectric point lower than that of the original ConA by 2.2 +/- 0.6 had the strongest binding activity for ovalbumin.