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.     

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.     

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.     

Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation

On-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed using a microinjector as the interface for performing two-dimensional (2-D) protein/peptide separations of complex protein mixtures. The focusing effect of CIEF not only contributes to a high-resolution protein/peptide separation, but also may permit the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. The preparative capabilities of CIEF are much larger than most of capillary-based electrokinetic separation techniques since the entire capillary is initially filled with a solution containing proteins/peptides and carrier ampholytes for the creation of a pH gradient inside the capillary. The focused peptides which have a similar pI are coinjected into the second separation dimension and further resolved by their differences in hydrophobicity. The resolving power of combined CIEF-CRPLC system is demonstrated using the soluble fraction of Drosophila salivary glands taken from a period beginning before steroid-triggered programmed cell death and extending to its completion. The separation mechanisms of CIEF and CRPLC are completely orthogonal and the overall peak capacity is estimated to be around approximately 1800 over a run time of less than 8 h. Significant enhancement in the separation peak capacity can be realized by further increasing the number of CIEF fractions and/or slowing the solvent gradient in CRPLC, however, at the expense of overall analysis time. The results of our preliminary studies display significant differences in the separation profiles of peptide samples obtained from salivary glands of animals staged at the 6 and 12 h following puparium formation.     

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.     

Comparison of different capillary isoelectric focusing methods--use of "narrow pH cuts" of carrier ampholytes as original tools to improve resolution

Two capillary isoelectric focusing (CIEF) systems have first been optimized: one uses a bare silica capillary and 30% (v/v) of glycerol in the separation medium while the other uses a coated capillary and an aqueous background electrolyte. To perform permanent capillary coating, two neutral polymers have been compared: hydroxypropylcellulose (HPC) and polyvinylalcohol (PVA). HPC coating gave best results for electroosmotic flow (EOF) limitation on a wide pH range: as compared to a bare silica capillary, it allowed to decrease EOF by 96% at pH 7.2 after acidic and basic treatments, whereas PVA coating lead only to a 76% decrease. The glycerol CIEF system was more satisfying for the separation of model proteins classically used as pI markers. Finally, the use of "narrow pH cuts" of carrier ampholytes added to commercial ampholyte mixtures allowed increasing resolution up to a factor 2.4 at a chosen pH for the separation of pI markers and milk proteins.     

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.     

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.     

Low‐molecular‐weight color pI markers to monitor on‐line the peptide focusing process in OFFGEL fractionation

High‐throughput mass spectrometry‐based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low‐molecular‐weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24‐wells device encompassing the pH range 3–10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI‐TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on‐line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom‐up proteomic approach with or without iTRAQ labeling.     

Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in-gel digestion and low-molecular-mass pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in-gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI-TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time-consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct pI values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post-translational modifications in prefractioned samples, where post-translational modifications (e.g., glycation) are frequent.     

Protein profiling by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

An automated system for intact protein analysis is described that combines capillary isoelectric focusing (CIEF), reversed-phase liquid chromatography (RPLC), and electrospray ionization-mass spectrometry (ESI-MS). Performance is demonstrated with a complex yeast enzyme concentrate. CIEF is performed with a microdialysis membrane-based cathodic cell that permits pI fractions to be sampled and stored for subsequent LC-MS analysis. A total of 50 microg protein is loaded onto the capillary. Ten fractions are stored which span the pI range 3-10. Each fraction is subsequently cleaned on a reversed-phase trap column and then characterized by LC-MS. MaxEnt1 is used to deconvolute the raw mass spectra to obtain the molecular weight (MW) of intact proteins/peptides in the sample. A two-dimensional display of pI vs. MW is illustrated for the 500 most prevalent species as identified by MaxEnt1.     

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.     

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.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

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.     

Capillary isoelectric focusing--reproducibility and protein adsorption

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3-15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long-time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self-amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)-coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused-silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.     

Evaluation of capillary isoelectric focusing in glycerol-water media with a view to hydrophobic protein applications

Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.     

A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B).     

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 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.