Protein analysis using capillary electrophoresis coupled to mass spectrometry through vibrating sharp-edge spray ionization.

Capillary electrophoresis (CE) interfaced to mass spectrometry (MS) with electrospray ionization typically incorporates acidic additives or organic solvents to assist in ionization. Vibrating sharp-edge spray ionization (VSSI) is a voltage-free method to interface CE and MS that does not require these additives, making it appealing for protein analyses. CE-VSSI nanoflow sheath separations are performed with low ionic strength aqueous solutions in the sheath to reduce suppression.

Fluorescent parallel electrophoresis assay of enzyme inhibition.

Background: Enzyme inhibitors comprise the largest class of pharmaceutical compounds. The discovery and development of new enzyme inhibitor drug candidates depends on sensitive tools to quantify inhibition constants, K, for the most promising candidates. A high throughput, automated, and miniaturized approach to measure inhibition is reported.

A Cost-Effective Microfluidic Device to Teach the Principles of Electrophoresis and Electroosmosis.

Electrophoresis is integral to analytical and biochemistry experiences in undergraduate education; however, fundamental principles of the method are often taught in upper-level laboratories through hands-on experiences. A laboratory activity is reported that teaches the concepts of electrophoretic mobility and electroosmotic flow. A single reuseable instrument, called a mini-E, costs 37 USD and consists of a DC power supply, a voltmeter, platinum electrodes, and a chip cast in polydimethylsiloxane.

Gels in Microscale Electrophoresis.

Gel matrices are fundamental to electrophoresis analyses of biopolymers in microscale channels. Both capillary gel and microchannel gel electrophoresis systems have produced fundamental advances in the scientific community. These analytical techniques remain as foundational tools in bioanalytical chemistry and are indispensable in the field of biotherapeutics.

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis.

Neuraminidase inhibitors modulate infections that involve sialic acids, making quantitative analyses of this inhibitory effect important for selecting and designing potential therapeutics. An automated nanogel capillary electrophoresis system is developed that integrates a 5 nL enzyme inhibition reaction in line with a 5 min separation-based assay of the enzymatic product to quantify inhibition as the half maximal inhibitory concentration (IC) and inhibitor constant (). A neuraminidase enzyme from is non-covalently immobilized in a thermally tunable nanogel positioned in the thermally controlled region of the capillary by increasing the capillary temperature to 37 °C.

A Low-Cost and Simple Demonstration of Freezing Point Depression and Colligative Properties with Common Salts and Ice Cream.

A laboratory activity was developed to teach freezing point depression and colligative properties to introductory-level chemistry students. The laboratory uses food-grade reagents and is delivered in two units that can be taught in a single 2 hour session or two separate sessions. The total cost of the consumables is 1 USD.

Nanoflow Sheath Voltage-Free Interfacing of Capillary Electrophoresis and Mass Spectrometry for the Detection of Small Molecules.

Coupling capillary electrophoresis (CE) to mass spectrometry (MS) is a powerful strategy to leverage a high separation efficiency with structural identification. Traditional CE-MS interfacing relies upon voltage to drive this process. Additionally, sheathless interfacing requires that the electrophoresis generates a sufficient volumetric flow to sustain the ionization process.

Simple, rapid, and reproducible capillary gel electrophoresis separation and laser-induced fluorescence detection of DNA topoisomers with unmodified fused silica separation capillaries.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA.

Capillary Nanogel Electrophoresis for the Determination of the β1-4 Galactosyltransferase Michaelis-Menten Constant and Real-Time Addition of Galactose Residues to -Glycans and Glycoprotein.

A thermally reversible nanogel is used in capillary electrophoresis to create discrete regions for a galactosyltransferase reaction and separation. The β1-4 galactosyltransferase enzyme, donor, and co-factor were patterned in the capillary. The substrate was driven through these zones and converted to galactosylated products, which were separated and identified.

Protein Sieving with Capillary Nanogel Electrophoresis.

Protein sieving, which is a fundamental tool in the biotechnology field, can be automated using capillary gel electrophoresis. The high-viscosity and biocompatible linear gels required for capillary sieving must be replaced for each run using high pressures. Thermally responsive gels are easier to renew in the capillary as they can be repetitively switched between low- and high-viscosity solutions.

Low Flow Voltage Free Interface for Capillary Electrophoresis and Mass Spectrometry Driven by Vibrating Sharp-Edge Spray Ionization.

Capillary electrophoresis-mass spectrometry is a powerful technique for high-throughput and high efficiency separations combined with structural identification. Electrospray ionization is the primary interface used to couple capillary electrophoresis to mass analyzers; however, improved designs continue to be reported. A new interfacing method based on vibrating sharp-edge spray ionization is presented in this work to overcome the challenges of decoupling applied voltages and to enhance the compatibility with separations performed at near-neutral pH.

Quantification of the α2-6 Sialic Acid Linkage in Branched N-Glycan Structures with Capillary Nanogel Electrophoresis.

Sialylation and sialic acid linkage in N-glycans are markers of disease but are analytically challenging to quantify. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated N-glycans in real time in the capillary so that N-glycan structures containing α2-6-linked sialic acid are easily separated, detected, and quantified. In this study, N-glycans were sequentially cleaved by enzymes at the head of the separation capillary so that the presence of α2-6-linked sialic acids corresponded to a shift in the analyte migration time in a manner that enabled interpretation of the N-glycan structure.

Profiling the N-Glycan Composition of IgG with Lectins and Capillary Nanogel Electrophoresis.

Glycosylated human IgG contains fucosylated biantennary N-glycans with different modifications including N-acetylglucosamine, which bisects the mannose core. Although only a limited number of IgG N-glycan structures are possible, human IgG N-glycans are predominantly biantennary and fucosylated and contain varying levels of α2-6-linked sialic acid, galactose, and bisected N-acetylglucosamine. Monitoring the relative abundance of bisecting N-acetylglucosamine is relevant to physiological processes.

Enhancing research for undergraduates through a nanotechnology training program that utilizes analytical and bioanalytical tools.

Nanotechnology is a broad field combining traditional scientific disciplines; however, analytical chemistry plays an important role in material design, synthesis, characterization, and application. This article emphasizes the uniqueness of nanotechnology and the importance of providing high-quality undergraduate research experiences to both attract and retain talented individuals to the field of nanotechnology. In response to this need to develop a strong and sustainable nanotechnology work force, strategies to create authentic research experiences are considered within the framework of an interdisciplinary nanotechnology environment at West Virginia University.

Capillary electrophoresis analysis of affinity to assess carboxylation of multi-walled carbon nanotubes.

Surface oxidation improves the dispersion of carbon nanotubes in aqueous solutions and plays a key role in the development of biosensors, electrochemical detectors and polymer composites. Accurate characterization of the carbon nanotube surface is important because the development of these nano-based applications depends on the degree of functionalization, in particular the amount of carboxylation. Affinity capillary electrophoresis is used to characterize the oxidation of multi-walled carbon nanotubes.

Capillary Electrophoresis Separations of Glycans.

Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. The method provides high resolution capable of separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on N-glycans, which are highly relevant to biological therapeutics and biomarker research.

Advances in enzyme substrate analysis with capillary electrophoresis.

Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for K determinations ranging from nanomolar to millimolar concentrations.

Peptide Probe for Multiwalled Carbon Nanotubes: Electrophoretic Assessment of the Binding Interface and Evaluation of Surface Functionalization.

Noncovalent interactions of peptides and proteins with carbon nanotubes play a key role in sensing, dispersion, and biocompatibility. Advances in these areas require that the forces which contribute to physical adsorption are understood in order that the carbon nanotubes present a degree of functionalization appropriate to the desired application. Affinity analyses of peptides are employed to evaluate the role of tryptophan and arginine residues in physical adsorption to carboxylated multiwalled carbon nanotubes.

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1-3)-linked galactose in glycans.

A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary.

Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K) for different substrates and ultimately provides a means to quantify the linkage (i.

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection.

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B.

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking.

Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles.

Quantification of circulating steroids in individual zebrafish using stacking to achieve nanomolar detection limits with capillary electrophoresis and UV-visible absorbance detection.

Capillary electrophoresis and UV-visible absorbance detection are used with sample stacking to achieve detection limits ranging from 0.2 to 2 ng/mL (0.8 to 6 nM) for steroids.