Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation (original) (raw)
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ELECTROPHORESIS, 2003
High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 mm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed highthroughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.
Divergent-flow isoelectric focusing for separation and preparative analysis of peptides
ELECTROPHORESIS, 2012
A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.
Journal of Chromatography A, 2009
Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. -Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H + and OH − ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.
Mixed-mode open tubular column for peptide separations by capillary electrochromatography
Mixed-mode chromatography open tubular column has been developed for peptide separation in electrochromatography. A column with 92 cm effective length and 50 μm internal diameter is fabricated internally with a copolymer sheet of restricted thickness. Catalyst facilitated binding of the coupling agent 3,5-bis (trifluoromethyl) phenyl isocyanate has been carried out at the interior surface of the column. The initiator sodium diethyldithiocarbamate was bound to the coupling agent. A small amount of N-[2-(acryloylamino) phenyl] acrylamide was used along with methacrylic acid and styrene in the monomer mixture to induce a little polar character in the stationary phase fabricated inside the column. Twentythree peptides have been separated from a chemically digested protein mixture present in cytochrome C in capillary electrochromatography, in addition to the separation of six commercial peptides. We achieved an average plate count of over 1.5 million/m with the column of current study both for the digested protein components and commercial peptides using 70/30% v/v (acetonitrile/20 mM ammonium formate) at pH 6.5. In addition, the column resulted in baseline separation of all the peptides with very good resolution, enhanced peak capacity, and better retention time span.
ELECTROPHORESIS, 2008
Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di-to decapeptides, and phosphinic pseudopeptides -tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH 2 -moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.
1999
High-resolution capillary electrophoretic separation of proteins and peptides was achieved by coating the inner wall of 75 pm ID fused-silica capillaries with 40-140 nm polystyrene particles which have been derivatized with a-a-diamines such as ethylenediamine or 1,lO-diaminodecane. A stable and irreversibly adsorbed coating was obtained upon deprotonation of the capillary surface with aqueous sodium hydroxide and subsequent flushing with a suspension of the positively charged particles. At pH 3.1, the detrimental adsorption of proteins to the capillary inner wall was suppressed efficiently because of electrostatic repulsion of the positively charged proteins from the positively charged coating which enabled protein separations with maximum efficiencies of 400 000 plates per meter. A substantial improvement of separation efficiency in particle-coated capillaries was observed after in-column derivatization of amino functionalities with 2,3-epoxy-l-propanol, resulting in a more hydrophilic coating. Five basic and four acidic proteins could be separated in less than 7 min with efficiencies up to l 900 000 theoretical plates per meter. Finally, coated capillaries were applied to the high-resolution analysis of protein glycoforms and bioactive peptides.
Protein fractionation in a multicompartment device using Off‐Gel™ isoelectric focusing
…, 2003
A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (pI) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with pI protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of b-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications. General 4 P. E. Michel et al.
Journal of Chromatography A, 1998
Separation conditions for antibodies, glycoproteins and peptides were optimized to fully realize the potential of automated imaged capillary isoelectric focusing (imaged cIEF) for protein analysis. Two commercially available capillary coatings, polyacrylamide and fluorocarbon, were found to provide reproducible results for cIEF separations. Both coatings could last 1 more than 100 runs under normal cIEF conditions. Up to 30 mM salts (Na ) could be added to samples to prevent protein precipitation before and during isoelectric focusing performed under imaged cIEF. Short analysis time of the imaged cIEF also aided in the prevention of protein precipitation. High current at the beginning of the focusing for samples in salt could be avoided by applying a voltage gradient. Additions of up to 6 M urea and 20% glycerol could enhance solubility of proteins and peptide. Imaged cIEF was applied to the quantitation of monoclonal antibodies.