Cheng S. Lee

On-line partial filling micelllar electrokinetic chromatography-electrospray ionization mass spectrometry

On-line combination of partial filling micellar electrokinetic chromatography (PF-MEKC) and electrospray ionization mass spectrometry (ESI-MS) is demonstrated for the analysis of triazine herbicides including atrazine, propazine, ametryne and prometryne. In comparison with conventional micellar electrokinetic chromatography (MEKC), PF-MEKC involves filling a small portion of the capillary with a sodium dodecyl sulfate (SDS) micellar solution for achieving the separation. In PF-MEKC, the triazine analytes first migrate into the micellar plug where the separation occurs and then into the electrophoresis buffer which is free of surfactant. Consequently, the electroosmotic transfer of neutral triazine herbicides to ESI-MS at the end of PF-MEKC capillary is comparable to conventional capillary zone electrophoresis ESI-MS. Therefore, PF-MEKC-ESI-MS provides a mechanism for the separation and mass detection of neutral molecules without the interference of surfactant.

High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis

On-line capillary isoelectric focusing (CIEF)-electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of hemoglobin variants A, C, S, and F. The effects of moving ionic boundary inside the CIEF capillary and MS scan rate on the separation resolution and mass detection of hemoglobin variants are investigated. The formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. Hemoglobin variants F and A, with a pI difference of 0.05 pH unit, are almost baseline resolved and identified in CIEF-ESIMS. The concentration detection limit for each hemoglobin variant is in the range of 10(-)(8) M, comparable to that obtained in two-dimensional gel electrophoresis using silver staining. Initial preconcentration during the focusing step and the use of single-ion monitoring scan mode are responsible for improving detection limits.

Micellar electrokinetic chromatography-mass spectrometry

The combination of micellar electrokinetic chromatography (MEKC) with mass spectrometry (MS) is very attractive for the direct identification of analyte molecules, for the possibility of selectivity enhancement, and for the structure confirmation and analysis in a MS-MS mode. The direct coupling of MEKC with MS can be hazardous due to the effect of nonvolatile MEKC surfactants on MS performance, including the loss of analyte sensitivity and ion source contamination. The possibility of off-line coupling between MEKC and matrix-assisted laser desorption/ionization (MALDI)-MS remains to be investigated. Various approaches for on-line coupling MEKC with electrospray ionization (ESI)-MS, including the use of high-molecular-mass surfactant, an electrospray-chemical ionization (ES-CI) interface, a voltage switching and buffer renewal system, partial-filling micellar plug and anodically migrating micelles, are reviewed and evaluated. The use of an ES-CI interface is most promising for routine operation of on-line MEKC-MS under the influence of nonvolatile salts and surfactants. The use of a high-molecular-mass surfactant allows the formation of a micellar phase at very low surfactant concentrations and avoids the generation of a high level of background ions in the low m/z region. Alternatively, the application of a partial-filling micellar plug and anodically migrating micelles eliminate the introduction of MEKC micelles into the ESI-MS system. It is possible to directly transfer the conventional MEKC separations to partial-filling MEKC-ESI-MS and MEKC-ESI-MS using anodically migrating micelles without any instrument modifications.

Monitoring the refolding pathway for a large multimeric protein using capillary zone electrophoresis

Abstract Rapid identification of transient partially folded intermediates formed during protein refolding and aggregation has been difficult, particularly with separation methods relying on solid matrices. Capillary zone electrophoresis equipped with laser-induced fluorescence detection provides a fast sensitive means of identifying folding and aggregation intermediates using the intrinsic tryptophan fluorescence. The in vitro refolding of the trimeric P22 tailspike, a model system for the study of protein folding, misfolding and aggregation, has been monitored after dilution out of denaturant. Both monomeric and trimeric folding intermediates were resolved. The refolding kinetics and yields measured by capillary zone electrophoresis were in good agreement with those obtained via fluorescence spectrophotometry and polyacrylamide gel electrophoresis. In comparison with typical UV detection, laser-induced tryptophan fluorescence increased detection sensitivity. In addition, the fluorescence signal carries information on the packing of the tryptophan residues in the folding intermediates. For tailspike and many other proteins, the off pathway aggregation reactions proceed from a thermolabile intermediate at the junction with the productive pathway. By monitoring refolding intermediates after temperature shifts, the structured monomeric intermediate was identified as the thermolabile junctional intermediate between the productive and aggregation pathways.

Effects of electroosmotic flow on zone mobilization in capillary isoelectric focusing.

The electroosmotic mobilization of focused protein zones in a fused-silica capillary is investigated using a mixture of model proteins, including alpha-chymotrypsinogen A (bovine pancreas), myoglobin (horse heart) and carbonic anhydrase II (bovine erythrocytes). The presence of carrier ampholytes in the entire capillary and the adsorption of carrier ampholytes onto the capillary wall almost eliminate the electroosmotic flow in the fused-silica capillary, obviating the need for polymer additives such as methylcellulose and hydroxypropylmethylcellulose. In fact, the electroosmotic displacement of focused protein zones can only be achieved by injecting a mixture of proteins and ampholytes as a plug at the inlet of a capillary that has been pre-filled with the catholyte. Various approaches for protein mobilization in the uncoated capillary completely filled with carrier ampholytes are studied. The addition of methylcellulose to the sample mixture of carrier ampholytes and protein analytes serves as an anticonvective medium during the gravity mobilization step and contributes to the reduction of protein adsorption onto the capillary wall.

Comparison of Protein Separations in Capillary Zone Electrophoresis and Capillary Isoelectric Focusing Interfacing with Electrospray Mass Spectrometry

On-line capillary zone electrophoresis/electrospray ionization mass spectrometry (CZE-ESMS) and capillary isoelectric focusing/electrospray ionization mass spectrometry (CIEF/ESMS) were employed for protein analysis. The separation mechanisms and the detection limits of CZE/ESMS and CIEF/ESMS were compared by using model proteins including cytochrome c (horse heart), ribonuclease A (bovine pancreas), myoglobin (horse heart), carbonic anhydrase I (human erythrocytes) and β-lactoglobulin A (bovine milk). The effect of a moving ionic boundary inside the electrophoresis capillary on the separation resolution of model proteins by CZE/ESMS and CIEF/ESMS is described. In CZE/ESMS, the formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be eliminated by using a common counterion in both the background electrolyte and the sheath liquid. Additionally, the moving ionic boundary in CIEF/ESMS is minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. The concentration detection limits of model proteins in a full-scan CIEF/ESMS analysis are in the region of 10 -7 M, ∼20-50 times lower than that achievable using CZE/ESMS. Sample preconcentration taking place during the focusing step in CIEF is responsible for the improved detection limits.

On-line post-capillary affinity detection of immunoglobulin G subclasses and monoclonal antibody variants for capillary electrophoresis

Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-alpha 1-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.