Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization

On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin beta chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR. The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study.     

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.     

High-efficiency capillary isoelectric focusing of peptides

Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated approximately 500-fold, and peptides present at < 1 ng/ microL were detectable using conventional UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points (deltapI approximately 0.01) could be resolved, High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.     

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.     

Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.     

Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.     

Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis

An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of approximately 240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 microg. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 microm to 100 microm, the required protein loading is further decreased from 9.6 microg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.     

Capillary isoelectric focusing of proteins with liquid core waveguide laser-induced fluorescence whole column imaging detection

A capillary isoelectric focusing (CIEF) system with liquid core waveguide (LCW) laser-induced fluorescence whole column imaging detection was developed in this study. A Teflon AF 2400 capillary was used as both the separation channel and the axially illuminated LCW. The excitation light was introduced at one end of the capillary, and propagated forward within the capillary. As the Teflon AF 2400 capillary has a refractive index (n = 1.29-1.31) lower than that of water (n = 1.33), total internal reflection was very apparent The employment of the Teflon AF 2400 capillary avoided the use of high refractive index additives such as glycerol, accommodating the system to wider applications. Due to its inert chemical properties, the capillary exhibited limited protein adsorption and electroosmotic flow; thus, the need for capillary preconditioning with polymeric solution and the addition of polymeric additives into the sample mixture can be avoided. Three types of proteins, naturally fluorescent proteins, covalently labeled proteins, and noncovalently labeled proteins, were examined using this method. CIEF under denaturing conditions was also explored, and several advantages over the native mode were found. When compared to a commercially available instrument with UV detection, the separation efficiency and peak capacity were similar while the detection sensitivity was enhanced by 3-5 orders of magnitude.     

UV embossed polymeric chip for protein separation and identification based on capillary isoelectric focusing and MALDI-TOF-MS

This paper demonstrates a ultraviolet (UV)-embossed polymeric chip for protein separation by capillary isoelectric focusing (CIEF) and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The polymeric chip was replicated by a UV-embossing technique using a soft rubber mold. Five diverse widely investigated families of UV-cured formulations were examined for MALDI ionization efficiency of bovine serum albumin (BSA) samples spotted on the polymer surfaces. The signal and signal-to-noise ratio from the polyester formulation were each 12 times those obtained with PMMA (a commonly used polymer material for MALDI) at picomole sample concentration. A polyester chip was successfully used to carry out CIEF to separate proteins, followed by MALDI-TOF-MS identification. Issues related to the successful chip fabrication and protein separation and identification are discussed.     

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.     

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.     

Coupling of solid-phase microextraction and capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection for protein analysis

A coupling method of solid-phase microextraction (SPME) and capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) was developed for the analysis of proteins. Unlike other liquid-phase separation methods and conventional CIEF, proteins are focused into stationary bands within a pH gradient in CIEF-WCID. Thus, CIEF-WCID is the most compatible liquid-phase separation method for coupling with SPME, which can effectively resolve the problems associated with the slow desorption kinetics of SPME in a liquid phase. By combining SPME and CIEF-WCID, the desorption time can be as long as necessary, allowing complete desorption without any band broadening and analyte carryover. By using this method, R-phycoerythrin in water can be extracted by SPME in 10 min, and subsequently analyzed by CIEF-LIF-WCID within 20 min, providing a limit of detection of 3.5 x 10(-12) M (S/N = 3). The feasibility of the SPME-CIEF-LIF-WCID method was demonstrated by extracting and analyzing extracellular phycoerythrins in cultured cyanobacteria samples. Extracellular phycoerythrins at the nanomolar level were extracted and analyzed in 30 min, while avoiding the interference of the cyanobacteria cells.     

Gradient elution isotachophoresis for enrichment and separation of biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.     

Mechanistic study on analyte focusing by dynamic pH junction in capillary electrophoresis using computer simulation

Dynamic pH junction is an on-line preconcentration method in capillary electrophoresis (CE) based on electrokinetic focusing of weakly ionic analytes with in large sample volumes in a multisection electrolyte system. In this report, experiments and computer simulations were performed to gain a better insight of the analyte focusing mechanism when a dynamic pH junction was used. A computer program, SIMUL, was used to simulate the band-narrowing process of a group for phenol derivatives under optimized buffer conditions, which were compared with experimental results. Computer simulations revealed the formation of a sharp moving pH boundary within the sample zone causing efficient focusing of long plugs of weakly acidic analytes based on their pKa. These studies offered useful information for understanding the band-narrowing process by control of the depth and lifetime of the moving pH boundary as a function of analyte pKa, sample pH, and injection length. The change in pH of the sample within the capillary was also estimated by measuring the absorbances of an analyte at two different wave-lengths. Optimization of analyte focusing resulted in enhanced detection responses of about 60-450-fold in terms of peak heights for some phenol derivatives' relation to conventional injections. Dynamic pH junction represents a novel approach to control band dispersion (peak width) and selectivity (mobility) of specific analytes for high-resolution CE separations.     

Dynamic kinetic capillary isoelectric focusing: a powerful tool for studying protein-DNA interactions

A new method called dynamic kinetic capillary isoelectric focusing (DK-CIEF) is presented for the study of protein-DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein-DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants.     

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.     

An etched porous interface for on-line capillary electrophoresis-based two-dimensional separation system

The construction and evaluation of an on-column etched fused-silica porous junction for on-line coupling of capillary isoelectric focusing (CIEF) with capillary zone electrophoresis (CZE) are described. Where two separation columns were integrated on a single piece of fused-silica capillary through the etched approximately 4 to 5-mm length porous junction along the capillary. The junction is easily prepared by etching a short section of the capillary wall with HF after removing the polyimide coating. The etched section becomes a porous glass membrane that allows only small ions related to the background electrolyte to pass through when high voltage is applied across the separation capillary. The primary advantages of this novel porous junction interface over previous designs (in which the interface is usually formed by fracturing the capillary followed by connecting the two capillaries with a section of microdialysis hollow fiber membrane) are no dead volume, simplicity, and ruggedness, which is particularly well suited for an on-line coupling capillary electrophoresis-based multiple dimensional separation system. The performance of the 2D CIEF-CZE system constructed by such an etched porous junction was evaluated by the analyses of protein mixtures.     

Two-dimensional protein separation with advanced sample and buffer isolation using microfluidic valves

Methods are described to achieve more efficient multidimensional protein separation in a microfluidic channel. The new methods couple isoelectric focusing (IEF) with high ionic strength electrophoretic separations by active microvalve control in a microchip. Several experiments demonstrating independent 2D separation were performed, and critical parameters for optimal chip performance were identified, including channel passivation, electroosmosis control, and IEF linearity control. This strategy can be used for integration of different heterogeneous separation techniques, such as IEF, capillary electrophoresis, and liquid chromatography. This new device can be ideal for preseparation and preconcentration of complex biomolecule samples for a streamlined biomolecule analysis using mass spectrometry.     

Aptamer-modified monolithic capillary chromatography for protein separation and detection

A capillary chromatography technique was developed for the separation and detection of proteins, taking advantage of the specific affinity of aptamers and the porous property of the monolith. A biotinylated DNA aptamer targeting cytochrome c was successfully immobilized on a streptavidin-modified polymer monolithic capillary column. The aptamer, having a G-quartet structure, could bind to both cytochrome c and thrombin, enabling the separation of these proteins from each other and from the unretained proteins. Elution of strongly bound proteins was achieved by increasing the ionic strength of the mobile phase. The following proteins were tested using the aptamer affinity monolithic columns: human immunoglobulin G (IgG), hemoglobin, transferrin, human serum albumin, cytochrome c, and thrombin. Determination of cytochrome c and thrombin spiked into dilute serum samples showed no interference from the serum matrix. The benefit of porous properties of the affinity monolithic column was demonstrated by selective capture and preconcentration of thrombin at low ionic strength and subsequent rapid elution at high ionic strength. The combination of the polymer monolithic column and the aptamer affinities makes the aptamer-modified monolithic columns useful for protein detection and separation.