Capillary Isoelectric Focusing Separation Research Articles

Imaged capillary isoelectric focusing employing fluorocarbon and methylcellulose coated fused silica capillary for characterization of charge heterogeneity of protein biopharmaceuticals

AbstractIdentifying and characterizing charge heterogeneity is essential for the successful development and production of biopharmaceuticals. Imaged capillary isoelectric focusing technology based on isoelectric point differentiation has been becoming the gold standard of quality and manufactory process control in the biopharmaceutical industry due to its high‐resolution characterization of protein charge variants with high throughput. Fluorocarbon‐coated capillaries are widely used in the imaged capillary isoelectric focusing to suppress the electroosmotic flow and minimize the protein adsorption onto the fused silica capillary inner surface. Additionally, polymers such as methylcellulose are usually added to the sample solution to serve as a dynamic capillary coating that improves the peak shape and resolution during the imaged capillary isoelectric focusing separation, especially for complex proteins. However, the addition of methylcellulose tends to result in tedious operation, “spike peaks” from bubble generation, and frequent capillary blockage during the imaged capillary isoelectric focusing separation. In addition, methylcellulose is not compatible with mass spectrometry and easily produces the contamination of mass spectrometry ion source when carrying out imaged capillary isoelectric focusing—mass spectrometry direct coupling. Recently, a new imaged capillary isoelectric focusing method was developed employing a methylcellulose‐coated capillary cartridge, to avoid the addition of methylcellulose which is then present throughout the whole analysis. When applied to protein drug characterization the established imaged capillary isoelectric focusing method demonstrated high repeatability, stable coatings, outstanding separation efficiency, and excellent isoelectric point differentiation. In addition, we compared imaged capillary isoelectric focusing separation using the methylcellulose‐coated capillary with that utilizing the routinely coated capillary such as fluorocarbon, illustrating that methylcellulose coating provided consistent results and could be seamlessly integrated into an existing drug discovery process. Finally, the methylcellulose‐coated fused silica capillary was applied to imaged capillary isoelectric focusing—mass spectrometry for characterizing protein charge variants allowing reliable identification of mass spectrometry after imaged capillary isoelectric focusing separation. This can greatly simplify the operation steps and prevent the contamination of mass spectrometry ion source that often results from using routinely coated capillaries ultimately making this an essential innovation of imaged capillary isoelectric focusing—mass spectrometry that greatly improves the imaged capillary isoelectric focusing compatibility with mass spectrometry.

High‐efficient characterization of complex protein drugs by imaged capillary isoelectric focusing with high‐resolution ampholytes

AbstractA carrier ampholyte is a molecule containing both acid and base functionality that is critical for imaged capillary isoelectric focusing. The quality of an imaged capillary isoelectric focusing separation for protein charge variants’ characterization is highly dependent on attributes of the carrier ampholytes used including baseline signal, linearity of the pH gradient, pI discrimination, and consistency between manufactured lots. AESlytes are a high‐resolution carrier ampholyte series that have been developed for the high‐resolution and selective characterization of diverse and complex protein drugs including diverse fusion proteins, antibody‐drug‐conjugate, bi‐specific antibodies, and viral proteins. While routine commercial ampholytes usually cannot solve such challenges, AESlytes demonstrate a reduction in baseline noise and distinguishably increased consistency between lots as compared to other commercial ampholytes. Here we apply AESlytes for the imaged capillary isoelectric focusing separation of several commercial fusion proteins and biosimilars with excellent repeatability. In addition, AESlytes with narrow‐range pH were employed directly coupled to a mass spectrometer for optimizing the separation resolutions allowing more reliable and accurate protein charge variant identification. Our study demonstrates that innovative high‐resolution carrier ampholytes as critical reagents play an essential role in high‐performance imaged capillary isoelectric focussing and tandem mass spectrometry analysis the routinely commercial ampholytes cannot achieve, especially for extremely complex protein drugs.

Evaluation of boronate affinity solid-phase extraction coupled in-line to capillary isoelectric focusing for the analysis of catecholamines in urine

In this study, a new miniaturized and integrated analytical system was developed based on the in-line coupling of boronate affinity solid phase extraction with capillary isoelectric focusing separation and UV detection. This original coupling takes advantage of the selective enrichment of cis-diol-containing compounds using a boronate affinity sorbent and the exceptional focusing features of isoelectric focusing process. Such coupling has been used for preconcentration/purification and separation of urinary catecholamines (dopamine, adrenaline and noradrenaline) as proof of concept. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column (8 cm) was chosen as solid phase extraction support due to its good chemical stability and its easy and versatile surface functionalization. Characterization of the miniaturized boronate affinity monolith column (μBAMC) was done by frontal affinity chromatography. An active-site amount of about 0.16 nmol cm−1 (75 μm i. d.) of phenyl boronic acid groups and Kd values ranging from 224 to 106 μM were obtained for catechol and catecholamines. A high loading volume (up to 15 times the affinity column volume) can be introduced with quantitative recovery yields. Optimization of the in-line coupling concerned the adaptation of (i) the μBAMC volume, (i.e. length and inner diameter of the monolithic column) for loading of large sample volumes and (ii) the CIEF experimental conditions. The ampholyte mixture was adapted (i.e. nature and concentration of carrier ampholytes, volume of sacrificial electrolytes) in order to ensure elution and separation of catecholamines and to decrease limit of detection down to 10–20 ng ml−1. The optimized method was applied to analyze urine samples.

Chip-capillary hybrid device for automated transfer of sample preseparated by capillary isoelectric focusing to parallel capillary gel electrophoresis for two-dimensional protein separation.

In this article, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to reroute the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed.

Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes

Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

Capillary Electromigration Separation of Proteins and Microorganisms Dynamically Modified by Chromophoric Nonionogenic Surfactant

A chromophoric nonionogenic surfactant poly(ethylene glycol) 3-(2-hydroxy-5-n-octylphenylazo)-benzoate, HOPAB, has been prepared and used as a buffer additive for a dynamic modification of proteins and/or microorganisms including Escherichia coli , Staphylococcus epidermidis (biofilm-positive and biofilm-negative), and the strains of yeast cells Candida albicans and Candida parapsilosis (biofilm-positive and biofilm-negative) during a capillary electrophoresis and a capillary isoelectric focusing (CIEF) with UV detection at 326 nm. Values of isoelectric points of labeled proteins and microorganisms have been calculated using UV-detectable pI markers and have been found comparable with pI of the native compounds. Minimum detectable amount has been assessed lower than picograms of proteins and lower than a hundred cells injected into a separation capillary. The introduced labeling method facilitates CIEF separation of microorganisms from the clinical sample of the infected urine at their clinically important levels in the pH gradient pH range of 2-5 and their subsequent cultivation. At the same time, it has enabled the determination of albumin in human urine as a major clinical marker of urinary tract infections and kidney diseases.

Validation of capillary zone electrophoresis and capillary isoelectric focusing separations optimized for the characterization of two recombinant products of the birch pollen allergen Bet v 1a

CZE and CIEF separation systems, both developed previously for a quality control of two recombinant products of the major birch pollen allergen Bet v 1a of Betula verrucosa, were validated including aspects of the International Conference on Harmonization. One product contained carbamylated variants as impurities. Linearity of response was confirmed by Mandel's fitting test between 0.028 and 1.90 mg/mL for CZE and between 0.016 and 0.26 mg/mL for CIEF. Repeatability and intermediate precision were evaluated for the effective mobility (mu(eff)) in CZE, for relative mobilization time in CIEF and the peak area ratio of Bet v 1a. LOQ for Bet v 1a was between 10 and 23 microg/mL for both methods. Evaluation of robustness for CZE revealed susceptibility of micro(eff) of Bet v 1a to alterations in of buffer pH and separation temperature. Selectivity was impaired by an increase in temperature, pH, and buffer concentration. In addition, pH variations influenced the separation profile of impurities. For CIEF, the ratio of narrow pH range carrier ampholytes is the critical parameter to retain robustness. Results demonstrate the suitability of both separation systems to discriminate between nonmodified Bet v 1a and carbamylated variants in the selected recombinant allergen products.

Capillary isoelectric focusing--mass spectrometry of proteins and protein complexes.

Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole-cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of noncovalent complexes on the intact complex level.

Integration of electrokinetic-based multidimensional separation/concentration platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis of Shewanella oneidensis.

This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring <500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.

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.

Comprehensive two dimensional separation based on coupling micellar electrokinetic chromatography with capillary isoelectric focusing.

Concentrating properties of the Capillary Isoelectric Focusing (CIEF) system with continuous whole-column-imaging detection were investigated for application as a second dimension in a comprehensive two-dimensional (2D) separation process. The concentration/separation/detection was completed within 4 min in a 300 microm inner diameter capillary. As the key to the successful coupling of CIEF to a first dimension separation, a novel interface was developed. A 10-port valve with two conditioning loops was used to perform both comprehensive collection and dialysis desalting of the first dimensional effluent, and as an interface coupling Micellar Electrokinetic Chromatography (MEKC) to CIEF. In the loop, salt and other unwanted first dimension effluent components were eliminated by dialysis and carrier ampholytes (CAs) were added. Peak broadening during the dialysis did not have significant impact on the CIEF separation because of its concentrating effect. Protein digests were first separated by MEKC followed by isoelectric point (pI) using whole-column-imaged CIEF. The dialysis interface allows general coupling of the whole-column-imaged CIEF to microscale separations.

On-target fraction collection for the off-line coupling of capillary isoelectric focusing with matrix-assisted laser desorption/ionization mass spectrometry.

In the present work, we describe a collection system for the off-line coupling of capillary isoelectric focusing (CIEF) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this system, the capillary effluent is directly deposited in fractions onto the MALDI target via the use of a sheath liquid. The collected fractions are subsequently supplemented with matrix and further analysed by MALDI-TOF mass spectrometry for mass assignment. The experimental set-up includes a fiber optic based UV detector operating at 280 nm, which allows the study of the influence of the sheath liquid composition on the CIEF separation. The influence of the carrier ampholyte concentration on the protein MALDI spectra was also evaluated and the feasibility of the collection method was finally demonstrated with a mixture of four standard proteins.

Capillary Isoelectric Focusing Separation Combined with Mass Spectrometry Using Sonic Spray Ionization for Protein Analysis.

Capillary isoelectric focusing (CIEF) separation combined with mass spectrometry for protein analysis has been demonstrated by using a sonic spray ionization (SSI) interface, which has a buffer reservoir between the ion source and the separation capillary. Since the buffer reservoir was used as an outlet reservoir for CIEF, on-line and one-step CIEF/MS was possible. In SSI, since a sample solution is sprayed at any solution-flow rate from a sample-introduction capillary with a high-speed gas flow, high-polymer ampholytes for CIEF can be used without clogging up the spray nozzle of the interface. Also, additives such as ampholytes significantly decrease ion intensity of analyte molecules; however, the SSI interface whose reservoir is filled with an acetic acid solution minimizes the decrease in ion intensity of the analyte. We combined CIEF with an ion trap mass spectrometer and were able to detect 160 fmol of myoglobin and cytochrome-c.

Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level.

The post-genomic era and increased demands for broad proteome measurements have greatly increased the needs for protein identification. We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae. Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D(10)) had been added. The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum. The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein. Proteins can then often be unambiguously identified based on their accurately determined molecular mass and the additional constraint provided by number of Leu residues. The identities of proteins were further confirmed by repeating CIEF/FTICR measurements with samples that contain other isotopically labeled amino acid residues (e.g. His, Arg, Ile, Phe, Lys). A theoretical study of the amino acid composition (for a difference in the amino acid sequence) showed the constraints needed in order to identify the protein unambiguously. Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications.

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.

Dynamics of capillary isoelectric focusing in the absence of fluid flow: high-resolution computer simulation and experimental validation with whole column optical imaging.

A 150-component, dynamic electrophoresis simulator was developed and applied to the description of capillary isoelectric focusing (CIEF) of amphoteric substances in quiescent solution. The simulator is shown to be capable of producing high-resolution pH 3-10 focusing data with 140 individual carrier ampholytes (20/pH unit) and at current densities that are used in CIEF, i.e., under conditions that were hitherto unaccessible by dynamic computer simulation. Having a focusing capillary of 5-cm length, the predicted focusing dynamics for amphoteric dyes obtained at a constant voltage of 1500 V (300 V/cm) are shown to qualitatively agree with data obtained by whole-column optical imaging. The simulation data provide detailed insight into the dynamics of the focusing process for the cases with the focusing column being sandwiched between 40 mM NaOH (catholyte) and 100 mM phosphoric acid (anolyte) or having the column ends only permeable for OH- and H+ at cathode and anode, respectively. Simulation data reveal that the number of sample boundaries migrating from the two ends of the column to the focusing positions is always equal to the number of sample components. The number of detectable migrating sample boundaries, however, can be lower. Whole-column optical imaging is demonstrated to be the method of choice for following the approach to equilibrium. With that detection format, transient sample peaks can be recognized and properly identified. This would also be possible with a scanning detector moving rapidly and repeatedly along the column but cannot be accomplished by a stationary detector placed at a specified location. The data presented demonstrate that the model together with imaging monitoring can be used to optimize the CIEF separation conditions.

Capillary Isoelectric Focusing without Carrier Ampholytes

A novel capillary isoelectric focusing (CIEF) separation of proteins in pure water was investigated using the whole-column imaging detection technique. Protons and hydroxyl ions are produced by the electrolysis of water, and the pH gradient is created by the electromigration of protons and hydroxyl ions into the separation capillary. The addition of acids to the anodic solution and bases to the cathodic solution enhances the formation of the gradients. Fast CIEF separation of proteins by electrolysis of water is demonstrated.

Capillary isoelectric focusing of yeast cells.

In the present work, capillary isoelectric focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (approximately 4-microm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-microm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (<3 cells/microL). CIEF experiments showed that yeast cell populations at early log, mid log, and stationary growth phases differ in isoelectric point, with values ranging from 5.2 to 6.4. The broader application of CIEF are projected for microorganism identification and separation based upon growth conditions.

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry.

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures.

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.