New Approaches in High Performance Capillary Electrophoresis of Biological Substances

Fused-silica capillaries having surface bound hydroxylated polyether functions were developed for the separation of proteins by capillary zone electrophoresis. In one approach, the hydrophilic coatings consisted of two layers; a glyceropropylpolysiloxane sublayer covalently attached to the inner surface and a polyether top layer. In a second approach, the capillary wall was coated with polysiloxane polyether chains whose monomeric units at both ends were covalently attached to the capillary inner surface with possible interconnection. These coatings yielded capillaries with different electroosmotic flow characteristics. The relatively long polyether chains of the various coatings were effective in shielding the unreacted surface silanols, thus minimizing solute-wall adsorption. As a consequence, high separation efficiencies were obtained in the pH range 4.0 to 7 .5, which allowed the separation of widely differing proteins, the characterization of heterogeneous proteins and the fingerprinting of crude protein mixtures. The various coatings were stable and exhibited reproducible separations from run-to-run, day-to-day, and column-to-column. Furthermore, a procedure was developed for restoring collapsed capillaries after prolonged use.A novel two-dimensional electrophoretic system for the control of electroosmosis in capillary zone electrophoresis has been developed and evaluated for rapid separations of proteins. The system comprises uncoated and polyether-coated fused silica capillaries coupled in series. An equation relating the average electroosmotic flow velocity in the coupled capillaries to the intrinsic electroosmotic velocities of the connected segments and their corresponding lengths has been derived and verified experimentally. This approach has the advantage of enabling the electroosmotic flow to be tuned independently of the applied voltage. As a consequence, rapid protein separations at relatively low field strength was achieved without sacrificing the high separation efficiencies obtained with surface modified capillaries.This article represents an extension to a: new approach, which was introduced very recently by our laboratory (see chapter 3), for the control of the magnitude of electroosmotic flow in capillary zone electrophoresis. In this new approach, short fused silica capillaries having different ~ potentials were coupled in series, and the amount of the electroosmotic flow was conveniently varied by changing the lengths of the individual capillary segments. The different coupled capillary systems evaluated in this study comprised various combinations of untreated fused silica capillaries and polyether coated capillaries having various electroosmotic flow characteristics. A general equation relating the average electroosmotic flow velocity in the coupled capillaries to the intrinsic electroosmotic velocity of the connected segments and their corresponding lengths has been derived and verified experimentally. The rate of the electroosmotic flow in a given system of coupled capillaries could be tuned over a range bordered by the lowest and highest intrinsic flow rates of the connected capillary segments. In addition, a system of coupled capillaries that permitted a stepwise change in the rate of the electroosmotic flow during analysis was introduced and evaluated. These elution schemes were useful in the rapid separation of oppositely charged proteins in a single electrophoretic run and in the rapid analytical determination of the various components of heterogeneous proteins.A new approach involving the stepwise increase in electroosmotic flow during analysis in capillary zone electrophoresis has been introduced and evaluated in the rapid separations of proteins and peptides. The stepwise increase in electroosmotic flow is based on the principle of coupled capillaries in series having different flow characteristics, a concept that was introduced recently by our laboratory. To produce stepwise changes in electroosmotic flow during analysis, a post-column multiple capillary device, which allowed the switching between several coupled capillary systems, was designed and constructed in-house. The utility of the multiple capillary device was also demonstrated and extended to fraction collection of separated analytes in short capillary segments. The fraction collection in capillaries facilitated the quantitative transfer of the collected fractions to high performance liquid chromatography (HPLC) for further analysis or to mass spectrometry (MS) for structural determination. The off-line combination of capillary zone electrophoresis with an HPLC or with a MS utilized commercial instruments without the need of expensive interfacing designs.Maltooligosaccharides derivatized with 2-aminopyridine were separated by capillary zone electrophoresis in the pH range 3.0 to 4.5 using 0.1 M phosphate solutions as the running electrolyte. The inclusion of small amounts of tetrabutylammonium bromide in the electrolyte solution facilitated the separation at pH 5.0 and yielded high separation efficiency. The separated zone of pyridylamino derivatives of maltooligosaccharides migrated across the fused silica capillary and passed the detection point in the order of increasing size. The "overall mobility" was a linear function of the number of glucose residues in the homologous series.Capillary zone electrophoresis with fused-silica tubes having hydrophilic coating on the inner walls was evaluated in the separation of peptide and glycopeptide fragments from trypsin digestion of a 1-acid glycoprotein. Submapping of glycosylated and nonglycosylated tryptic fragments of the glycoprotein by capillary electrophoresis was facilitated by selective isolation of the glycopeptides on concanavalin A silica-based stationary phases prior to the electrophoretic run. In addition, the electrophoretic map and submaps of the whole tryptic digest and its concanavalin A fractions, respectively, allowed the elucidation of the microheterogeneity of the glycoprotein. Also, capillary zone electrophoresis proved suitable for the mapping of the oligosaccharide chains cleaved from the glycoproteins by endoglycosidase digestion. The oligosaccharides cleaved from human and bovine a1-acid glycoprotein were analyzed after derivatization with 2- aminopyridine, which allowed their sensitive detection by on-column UV absorption. The separation was best achieved when 0.1 M phosphate solution, pH 5.0, containing 50 mM teterabutylammonium bromide was used as the running electrolyte. The effect of the organic salt on separation was attributed to ion-pair formation and/or hydrophobic interaction.The electrophoretic behavior of derivatized linear and branched oligosaccharides from various sources was examined in capillary zone electrophoresis with polyether coated fused-silica capillaries. Two UV absorbing (also fluorescent) derivatizing agents (2- aminopyridine and 6-aminoquinoline) were utilized for the electrophoresis and sensitive detection of neutral oligosaccharides, e.g., N-acetylchitooligosaccharides, high-mannose glycans and xyloglucan oligosaccharides. The oligosaccharides labelled with 6- aminoquinoline yielded eight times higher signal than those tagged with 2-aminopyridine. Plots of logarithmic electrophoretic mobilities of labelled N-acetylchitooligosaccharides with 6-aminoquinoline or 2-aminopyridine versus the number of sugar residues in the homologous series yielded straight lines in the size range studied, the slopes of which were independent of the tagging functions. The slopes of these lines are referred to as the Nacetylglucosaminyl group mobility decrement. The oligosaccharides were better resolved in the presence of tetrabutylammonium bromide in the running electrolyte. Furthermore, the electrophoretic mobilities of branched oligosaccharides were indexed with respect to linear homooligosaccharides, an approach that may prove valuable in correlating and predicting the mobilities of complex oligosaccharides.Enzymophoresis with coupled heterogeneous capillary enzyme reactors-capillary zone electrophoresis was developed and evaluated in the area of nucleic acids. Ribonuclease T1, hexokinase and adenosine deaminase were successfully immobilized on the inner walls of short fused-silica capillaries through glutaraldehyde attachment These open-tubular capillary enzyme reactors were quite stable for a prolonged period of use under operation conditions normally used in capillary zone electrophoresis. The capillary enzyme reactors coupled in series with capillary zone electrophoresis served as peak locator on the electropherogram, improved the system selectivity, and facilitated the quantitative determination of the analytes with good accuracy. Also, they allowed the online digestion and mapping of minute amounts of transfer ribonucleic acids, and the simultaneous synthesis and separation of nanogram quantities of oligonucleotides.Chemistr

Discrimination of glycoproteins via two-color laser-induced fluorescence detection coupled with postcolumn derivatization in capillary electrophoresis

Here, we report a novel method consisting of capillary electrophoretic separation followed by two-color LIF detection with postcolumn derivatization. The method can be used to discriminate glycoproteins in a protein mixture containing both glycosylated and unglycosylated proteins. The detector permitted simultaneous measurements of two electropherograms obtained by 450 nm (diode laser) and 532 nm (Nd:YAG laser) lasers excited native proteins following postcolumn derivatization with naphthalene-2,3-dicarboxaldehyde and concanavalin A (Con A) labeled with tetramethylrhodamine (rhodamine-labeled Con A), respectively. So, a protein can be assigned as glycosylated if it shows a peak at the same migration time in both electropherograms. According to the proposed principle, in a single run we discriminated a glycosylated protein (thyroglobulin) from an unglycosylated protein (albumin) in the presence of rhodamine-labeled Con A. Because the methodology permits the simultaneous detection of native proteins and their complexes with a fluorescently labeled probe, it should have broad applicability to binding assays

Glycan labeling strategies and their use in identification and quantification

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed