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.     

Sol-gel column technology for capillary isoelectric focusing of microorganisms and biopolymers with UV or fluorometric detection

The sol-gel surface modification is used for capillary isoelectric focusing of microorganisms and biopolymers. The coating strongly decreases the electroosmotic flow so that it enables the use of the short capillaries down to 100 mm in the separation length. The examples of capillary isoelectric focusing of the low-molecular-mass pI markers and mixed cultures of microbial populations of Escherichia coli, Candida albicans, Staphylococcus epidermidis, and Enteroccocus faecalis with UV detection are shown. It is possible to quantify bacterial cells according to their peak areas; the minimum detectable number of microbial cells was 5 x 10(2)-1 x 10(3). The compatibility of sol-gel capillaries with sensitive fluorometric detection of fluorescent pI markers together with fluorescein labeled proteins is demonstrated.     

Labeling effects on the isoelectric point of green fluorescent protein.

We studied the effects of fluorescent labeling on the isoelectric points (pI values) of proteins using capillary isoelectric focusing with laser-induced fluorescence detection (cIEF-LIF). Specifically, we labeled green fluorescent protein (GFP) from the jellyfish Aequorea victoria with the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). cIEF-LIF was used to monitor the native fluorescence of GFP and showed pI changes in GFP's FQ-labeled products. Multiple labeling of GFP with FQ produced a series of products with pI values shifted towards a low pH. We verified cIEF-LIF results with traditional slab gel IEF. Our cIEF-LIF technique can routinely detect 10(-11) M of FQ-labeled protein, whereas traditional slab gel IEF with silver stain detection gives detection limits of 10(-7) M in the same samples.     

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.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Pharmaceutical applications of isoelectric focusing on microchip with imaged UV detection

For the first time, the application of a commercial Shimadzu microchip electrophoresis system MCE-2010 equipped with an imaging UV detector for isoelectric focusing (IEF) of therapeutic proteins is reported. By proper adjustment of the pH gradient, samples with pI values ranging from 2.85 to 10.3 can be focused to the imaged part of the separation channel. Three therapeutic proteins (hirudin, erythropoietin, and bevacizumab) have been successfully focused on the microchip, and the results have been compared to conventional capillary IEF in terms of peak profile, pI values, and reproducibility.     

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.     

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.     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

Fluorescence detection system for capillary separations utilizing a liquid core waveguide with an optical fibre-coupled compact spectrometer

A fluorescence detection system for capillary liquid separation methods is described. The system is based on a silica capillary coated with a low refractive index fluoropolymer Teflon AF that serves both as a separation channel and as a liquid core waveguide (LCW). A fibre-coupled laser excites separated analytes in a detection point and arising fluorescence is collected at one end of the LCW capillary into the other optical fibre which brings it to a compact charge-coupled device (CCD) array spectrometer installed in a desktop computer. No additional components such as focusing optics or filters are necessary. This system was used for detecting isoelectrically focused fluorescent low-molecular-mass pI (isoelectric point) markers and fluorescein isothiocyanate (FITC) labelled proteins. The ability of the system to acquire fluorescent spectra is also demonstrated.     

Two-dimensional gel isoelectric focusing

Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90 degrees (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF. During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of beta-lactoglobulin (pI values 5.14 and 5.31, respectively) non-separated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF. Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.     

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.     

Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.06% and 0.95%, respectively, when using IgG(4) as a model. Furthermore, RSDs of charge variant compositions were less than 5.0%. These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution, and it is applicable to quality testing and detailed characterization in the pharmaceutical industry. In addition, it should be noticed that the method provided non-linear pH gradient within the tested ranges, from pI 9.50 to 3.78, and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF. This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values.     

Separation of phenotypically indistinguishable Candida species, C. orthopsilosis, C. metapsilosis and C. parapsilosis, by capillary electromigration techniques

At the current state of laboratory diagnostics, methods for fast identification of phenotypically indistinguishable species are difficult or inaccurate. An example is represented by Candida parapsilosis, which is the second most common yeast species isolated from bloodstream infections. C. parapsilosis comprises a complex of three genetically distinct groups. Genotypes II and III have been designated as the separate species Candida orthopsilosis and Candida metapsilosis, phenotypically indistinguishable. The considerable genetic variability of these newly described yeasts species has caused difficulties in the development of molecular techniques for their precise identification. Similarly, the detection of biofilm formation, which is considered as an important yeast virulence factor, is accompanied by difficulties. In this study we optimize the first precise and reproducible method for the separation and possible identification of C. orthopsilosis, C. metapsilosis and C. parapsilosis as well as the detection of their ability to form biofilm. The method is based on capillary isoelectric focusing and capillary electrophoresis with UV detection. In capillary isoelectric focusing, very narrow pH gradients were established. With such gradients, differences in isoelectric points of biofilm-negative and biofilm-positive species calculated from the migration times of the selected pI markers were below 0.03 pI units. In the capillary zone electrophoresis narrow zones of the cells of Candida species were detected with sufficient resolution. The values of the isoelectric point and the migration velocities of the examined species were independent on the origin of the tested strains. Capillary isoelectric focusing was examined also for the separation and detection of the cultivated biofilm-negative C. parapsilosis in the blood serum.     

CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection

The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.     

The use of immobilized pH gradients for the detection of human polymorphisms in the forensic identification of bloodstains.

Some polymorphic proteins (alpha 1-antitrypsin, orosomucoid, transferrin, group specific component, plasminogen) and enzymes (phosphoglucomutase, acid phosphatase, estrase D) were determined in bloodstain extracts by isoelectric focusing with carrier ampholytes (CA) and with immobilized pH gradients (IPGs) rehydrated with CA. IPGs yield superior results for typing of genetics markers in bloodstains since phenotypes are better distinguished and the bands are straighter and sharper in the presence of contaminants. Also, the sensitivity of IPGs with CA is similar to isoelectric focusing (IEF) with CA. A new variant, ACP*B1, found in Negroid west African populations and not found in Caucasians is described. Such a variant can only be determined by IPGs since its isoelectric point (pI 5.95) is close to that of the ACP*B (pI 6.05) variant.     

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.     

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.     

New isoelectric buffers for capillary electrophoresis: N-carboxymethylated polyethyleneimine as a macromolecular isoelectric buffer

Isoelectric buffers are attractive for electrophoresis because of their low conductivity, and their compatibility with indirect photometric detection in capillary electrophoresis (CE) where they do not interfere with the detection by exhibiting competitive displacement of the UV-absorbing probe ion. N-carboxymethylated polyethyleneimine (CMPEI) was prepared by introducing a half molar equivalent of carboxylate groups onto a polyethyleneimine backbone. Its isoelectric point determined by conductometric titration and from the pH of its dilute aqueous solution is approx. 6.8, which allows isoelectric buffering at a lower pH compared to histidine (pI7.7). Although the isoelectric point is somewhat diffuse, as expected for a polymeric compound, it exhibits a buffering capacity at a pI point of about twice that of histidine. Studies of electroosmotic flow (EOF) profile at various pH values in fused silica capillaries showed that CMPEI adsorbs onto the fused silica wall and reverses the EOF at pH < 6.5. CMPEI was applied as a buffer in an electrolyte containing 0.5 mM of the anionic dye tartrazine used as the probe for indirect detection of anions. The separation system exhibited a stable baseline, no system peaks, separation efficiencies of up to 195,000 theoretical plates, and detection limits down to 0.2 microM or 2 amol of injected analyte.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.