A microfabricated device has been developed in which electrospray ionization is performed directly from the corner of a rectangular glass microchip. The device allows highly efficient electrokinetically driven separations to be coupled directly to a mass spectrometer (MS) without the use of external pressure sources or the insertion of capillary spray tips. An electrokinetic-based hydraulic pump is integrated on the chip that directs eluting materials to the monolithically integrated spray tip. A positively charged surface coating, PolyE-323, is used to prevent surface interactions with peptides and proteins and to reverse the electroosmotic flow in the separation channel. The device has been used to perform microchip CE-MS analysis of peptides and proteins with efficiencies over 200 000 theoretical plates (1 000 000 plates/m). The sensitivity and stability of the microfabricated ESI source were found to be comparable to that of commercial pulled fused-silica capillary nanospray sources.
A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel where cellular constituents were separated and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the α and β subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute demonstrating the potential of this method for high-throughput single cell analysis.
Here, we demonstrate an integrated microfluidic capillary electrophoresis-electrospray ionization (CE-ESI) device for the separation of intact monoclonal antibody charge variants with online mass spectrometric (MS) identification. The need for dynamic coating and zwitterionic background electrolyte (BGE) additives has been eliminated by utilizing surface chemistry within the device channels to control analyte adsorption and electroosmotic flow (EOF) while maintaining separation efficiency. The effectiveness of this strategy was illustrated with the separation of charge variants of Infliximab. Three major species corresponding to C-terminal lysine variants were separated with an average resolution of 0.80 and identified by mass difference. In addition to the lysine variants, masses were determined for minor acidic and basic species. The separation of these variants prior to MS analysis facilitated the identification of glycosylation patterns for each of the variants. The general applicability of this method was demonstrated by analyzing two additional monoclonal antibody species: an IgG2 antibody and an IgG1 antibody conjugate. The IgG2 proved to have similar modifications to Infliximab with lower relative abundances of the lysine variants. Analysis of the IgG1 drug conjugate further exemplified the advantages of MS detection; differences in the extent of antibody conjugation were detectable despite limited CE resolution. The CE-ESI-MS methodology described here is a rapid and generic strategy for the separation of intact mAb charge variants and facilitates the identification of variants through MS detection.
A new ethyl-bridged hybrid packing material was evaluated in terms of its suitability for ultrahigh-pressure liquid chromatography (UHPLC). The 1.5-microm particles were obtained and packed into 30-microm-i.d. fused-silica capillary columns up to 50 cm in length. The particles were evaluated by isocratic reversed-phase UHPLC at pressures up to 4500 bar (65,000 psi). The chromatographic performance of these particles was found to be similar to the performance of 1.0-microm nonporous silica particles. The mechanical strength of the ethyl-bridged hybrid material was evaluated by running a 15-cm-long column at pressures up to 4500 bar. No breakdown of the particles in the packed bed was observed. The sample loading capacity of the hybrid material was evaluated and compared to 1.0-microm nonporous silica material by observing analyte peak width versus amount injected. The observed improvement in loading capacity for the hybrid material versus nonporous silica was consistent with the improvement predicted by comparing the phase ratios of the two materials.
A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analysis of protein digests were performed and compared.
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