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Glycoproteomic analysis requires efficient separation and sensitive detection to enable the comprehensive characterization of glycan heterogeneity. Here, we report the use of capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) with an electrokinetically-pumped nanospray interface for the study of protein glycosylation microheterogeneity. A fast separation was developed...
We demonstrate an electrokinetically pumped sheath flow nanospray interface for capillary electrophoresis coupled to negative mode electrospray mass spectrometry. In this interface, application of an electric field generates electro-osmotic flow at the interior of a glass emitter that is pulled to a 10–20µm inner diameter orifice. Electro-osmotic flow pumps liquid around the distal tip of the separation...
Capillary zone electrophoresis–electrospray ionization–mass spectrometry (CZE–ESI–MS) was used for analysis of reduced antibodies. We first developed a simple protocol to condition commercial linear-polyacrylamide coated capillaries for use in top–down proteomics. We then suspended reduced antibodies in a solution of 35% acetic acid, 50% acetonitrile in water. Heavy and light chains were baseline...
Proteomic analysis using capillary zone electrophoresis (CZE) typically is performed with linear polyacrylamide (LPA) coated capillaries. These capillaries both minimize the adsorption of peptides and proteins to the inner wall of the capillary and decrease electroosmosis, which increases the separation capacity. LPA coating protocols were first reported by Hjerten in 1985. Conventional LPA production...
A sulfonate–silica hybrid strong cation-exchange (SCX) monolith was synthesized at the proximal end of a capillary zone electrophoresis column and used for on-line solid-phase extraction (SPE) sample preconcentration. Sample was prepared in an acidic buffer and deposited onto the SCX-SPE monolith and eluted using a basic buffer. Electrophoresis was performed in an acidic buffer. This combination of...
A fraction collector based on a drop-on-demand ink-jet printer was developed to interface capillary zone electrophoresis with a 96 well microtiter plate. We first evaluated the performance of the collector by using capillary zone electrophoresis to analyze a 1mM solution of tetramethylrhodamine; a fluorescent microtiter plate reader was then used to detect the analyte and characterize fraction carryover...
Automated diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized enzyme reactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. Components undergo a preliminary separation in the first capillary. Fractions are parked in the reactor where some components undergo transformation. The...
A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored...
A rapid and reproducible system that couples capillary isoelectric focusing to a high-resolution mass spectrometer was developed for on-line analysis and identification of protein digests. Magnetic microsphere-based immobilized trypsin was used for protein digestion to reduce the digestion time to 10min, with a total analysis time of 4h. A three-protein-mixture (myoglobin, BSA, cytochrome c) with...
Capillary electrophoresis can provide fast and efficient separations of peptides. However, the high speed separation and limited loading capacity of capillary electrophoresis requires the use of a fast and sensitive detector. While laser-induced fluorescence provides exquisite sensitivity and millisecond response time, it inherently generates a low information content signal. In contrast, mass spectrometry...
A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection...
An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The...
Two-dimensional capillary electrophoresis was used for the separation of proteins and biogenic amines from the mouse AtT-20 cell line. The first-dimension capillary contained a TRIS–CHES–SDS–dextran buffer to perform capillary sieving electrophoresis, which is based on molecular weight of proteins. The second-dimension capillary contained a TRIS–CHES–SDS buffer for micellar electrokinetic capillary...
The design of a simple, highly miniaturized instrument for manual microsequence analysis of proteins and peptides is described. The reaction chamber is made of fused silica capillary tubing with all reagents and solvents necessary for coupling and cleavage delivered via two valves and a syringe-based dispenser. Only two pressure regulators are required. A section of the flow-through reaction chamber...
A miniaturized protein and peptide microsequencer consisting of a fused silica capillary reaction chamber is described. Extremely small volumes of reagents, 2 μl or less, were delivered directly to the reaction chamber through individual fused silica capillary lines using low pressure syringe pumps. Other than an argon gas controller, no valves were used in the delivery system. The short flow path...
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