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Intrinsic fluorescence imaging (IFI) has been used for the stain-free detection of proteins in slab gel. However, complicated detection setups and small irradiation area limited the development of facile, online, and portable imaging of the whole slab gel. We here designed a quadruple UV LED array to produce even and powerful area light for direct irradiation of gel electrophoresis chip (GEC) at 275 nm. In addition, we only used a filter of 365 nm, a UV camera lens, and a CCD for IFI detection. We integrated the simple detection setup with the small GEC to construct the IFI-GEC device with a portable size of 15 × 15 × 38 cm. We detected three model proteins to demonstrate the good evenness of the LED array and the online imaging of the whole GEC. Furthermore, the reproducible IFI-GEC detection was completed within 10 min and the LOD was as low as 40 ng for lysozyme detection. All results indicated the potential of the IFI-GEC device for online and portable detection of proteins without staining.

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.

Native protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for native proteins with molecular weights from 20,000 to 47,000. The separation of native proteins in gel-filled capillaries is based solely on the charge of the protein as in normal zonal electrophoresis. Retention of protein activity in the acrylamide matrix was demonstrated by performing enzymatic assays in the gel matrix. Alkaline phosphatase (ALP) and beta-galactosidase assays were conducted in both C18-PF108-modified and polyacrylamide gel-filled capillaries. Enzyme assays were achieved by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the reaction of enzyme with substrate was monitored using a standard UV-visible detector. Both constant potential and zero potential modes of analysis were demonstrated. The polyacrylamide gel columns provide the advantages of minimized diffusion and limited band spreading due to the high viscosity of the gel matrix. The lowest detection limit achieved was 5.2 x 10(-20) mol (7.6 x 10(-12) M sample injected) of ALP. The dual enzyme assay of ALP and beta-galactosidase was achieved in gel-filled capillaries simultaneously.

This paper demonstrates the use of UV-transparent replaceable polymer networks for the separation of SDS-protein complexes on the basis of molecular weight. First, the use of linear (i.e. non-cross-linked) polyacrylamide is shown to provide molecular separation of SDS-protein complexes. A study reveals such columns to yield significantly greater lifetime than cross-linked gels because of the flexibility of the noncovalently attached polymer chains. However, column lifetime was still found to be limited (approximately 20-40 injections), and detection at 214 nm was problematical because of the absorbance of polyacrylamide. UV-transparent polymer networks of dextran and PEG were substituted for polyacrylamide with successful molecular weight sieving of SDS-protein complexes at 214 nm. Due to their low to moderate viscosities, these networks could be routinely replaced leading to the possibility of hundreds of injections with a single column. Migration time reproducibilities of 0.5% RSD or less were found with replacement of the network. Using dextran, calibration plots of peak area vs concentration of standard protein were linear over the range of 0.5 microgram/mL up to at least 0.25 mg/mL. Furthermore, plasma samples could be directly utilized because of the strong solvating power of SDS. Rapid separation of protein mixtures are demonstrated with these UV-transparent polymer networks.

A simple method for the off-line coupling of laser desorption mass spectrometry (LDMS) and capillary zone electrophoresis (CZE) is described. Representative mass spectra of subpicomole quantities of proteins isolated from CZE are presented and discussed. The current detection limit for bovine alpha-lactalbumin is 100 fmols injected onto the CZE column. Horse heart myoglobin was demonstrated to be stable in CHES/KCl, a CZE buffer, for at least 1 month, suggesting that some isolates can be safely stored for long time periods prior to LDMS analysis. Protein stability in 0.1% aqueous trifluoroacetic acid (TFA), a common solvent for LDMS, must also be considered. In the special case of porcine pepsinogen, significant (greater than 50%) degradation was observed within 5 min in TFA. In favorable cases, mass measurement accuracies of +/- 0.02% were obtained for protein isolates. Factors limiting mass measurement accuracy are presented. Finally, the possibility of identifying protein isolates, by combining N-terminal sequencing, molecular mass measurements, and selective peptide "mapping" procedures, is discussed.

The free zone electrophoretic mobility of proteins can be predicted from the protein's amino acid content by applying a model based on the Debye-Hückle-Henry theory and Henderson-Hasselbalch equation. Calculated mobilities are always greater than actual mobility but a pH-independent proportionality (described by the constant FZ) is found between the two. Thus, determination of a protein's mobility at one pH allows, with the use of the model and FZ, calculation of its mobility at other pH conditions. This leads directly to optimum conditions for the electrophoretic resolution of proteins in capillary zone electrophoresis. The fundamental nature of FZ is examined and found to be a function of a proteins molecular weight, charge, and solution ionic strength. This work aids in explaining the form of previously proposed empirically based equations for peptide and protein mobility.

Capillary zone electrophoretic separations of proteins have been achieved by using nonionic surfactant coated capillaries. Capillaries were prepared by derivatization of the silica surface with octadecylsilane followed by the deposition of a layer of nonionic surfactant from an aqueous solution above the critical micelle concentration. This coating is of sufficient thickness and hydrophilicity to reduce both protein adsorption and electroosmotic pumping. This hydrophilic coating reduces electroosmotic pumping 5-8-fold while resolving proteins quickly and efficiently with good recovery. The coating provides a stable and reproducible means of deactivation, while the rate of electroosmotic pumping stays relatively constant throughout the pH range 4-11. This allows the pH to be varied to enhance selectivity without adversely affecting the flow rate.

A procedure for obtaining highly stable coated capillaries for use in capillary electrophoresis (CE) is described. Reaction of surface-chlorinated fused silica capillaries with the Grignard reagent, vinyl magnesium bromide, followed by reaction of the vinyl group with acrylamide, results in an immobilized layer of polyacrylamide attached through hydrolytically stable Si-C bonds. This method is an extension of the capillary coating procedure described previously by Hjerten, differing in the means by which the polyacrylamide layer is bonded to the capillary walls. Capillaries treated in the manner described here can be used over a pH range of 2-10.5, without noticeable decomposition of the coating. In comparison to uncoated capillaries, separations of proteins using such coated capillaries are improved due to a reduction in protein adsorption to the capillary walls, although interaction is still present to some degree as evidenced by an inability to obtain plate counts as high as those predicted by theory. Electroosmotic flow is virtually eliminated in the coated capillaries, resulting in improved reproducibilities of protein migration times in comparison to uncoated capillaries. Additionally, peak skew is evaluated for model proteins and improvements are noted for the coated capillaries. Results are presented for separations of model protein mixtures, comparing the performance of the vinyl-bound polyacrylamide coated capillaries and uncoated capillaries at both high and low pH extremes.

A mathematical model of the electrophoretic behavior of proteins is presented. The Debye-Hückel-Henry theory is used for the description of protein mobility, which has the important result of making net mobility a function of ionic strength. A net charge vs pH relationship and a diffusion coefficient are required to describe a specific protein. The model is employed for the computer simulation of three distinct electrophoretic modes: isoelectric focusing, isotachophoresis, and zone electrophoresis. The validity of the model is tested by comparing simulation with experimental data. Excellent qualitative agreement was found.

We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties.

Peptide mapping with mass spectrometry (MS) detection is a powerful technique routinely used for interrogating physicochemical properties of proteins. Peptide mapping benefits from an efficient front-end separation to increase selectivity and reduce complexity prior to MS detection. The most commonly used method for peptide mapping is based on reverse phase liquid chromatography with mass spectrometry. Capillary zone electrophoresis with mass spectrometry (CZE-MS) is an orthogonal technique with growing attention for peptide mapping of biotherapeutic proteins due to its high efficiency and sensitivity. However, that growth has been slow due to poorer peptide resolution and method robustness compared to RPLC. Here we present results from optimization of CZE-MS peptide mapping separation using mixed aqueous--aprotic dipolar solvent (N,N-dimethylacetamide (DMA) and N,N-dimethylformamide (DMF), as the background electrolyte (BGE) to improve the separation performance. Addition of DMA or DMF to the BGE impacts separation selectivity through differential change in pKa of the peptides. The CZE-MS peptide mapping method with the modified BGE produced significant improvement in resolution over the conventional CZE-MS methods. The method was evaluated with both sheathless and sheathflow CE-MS ion sources. [ABSTRACT FROM AUTHOR]

We present an origami paper-based electrophoretic device (oPAD-Ep) that achieves rapid (∼5 min) separation of fluorescent molecules and proteins. Due to the innovative design, the required driving voltage is just ∼10 V, which is more than 10 times lower than that used for conventional electrophoresis. The oPAD-Ep uses multiple, thin (180 μ/layer) folded paper layers as the supporting medium for electrophoresis. This approach significantly shortens the distance between the anode and cathode, and this, in turn, accounts for the high electric field (>1 kV/m) that can be achieved even with a low applied voltage. The multilayer design of the oPAD-Ep enables convenient sample introduction by use of a slip layer as well as easy product analysis and reclamation after electrophoresis by unfolding the origami paper and cutting out desired layers. We demonstrate the use of oPAD-Ep for simple separation of proteins in bovine serum, which illustrates its potential applications for point-of-care diagnostic testing. [ABSTRACT FROM AUTHOR]

Amphiphilic tris-urea molecules self-assemble into a supramolecular hydrogel in tris(hydroxymethyl)aminomethane--glycine buffer. The supramolecular hydrogel is used as a matrix for the electrophoresis of acidic native proteins, in which proteins are separated based on their isoelectric points rather than their molecular weights. The proteins remain in their native forms during migration, and their activities are retained after electrophoresis. Glucoside substituents on the amphiphilic tris-urea molecule allow for the affinity electrophoresis of a carbohydrate-binding protein to be performed. The proteins can be efficiently recovered from the supramolecular hydrogel using a simple procedure. This is a major advantage of using this noncovalent, self-assembled material. [ABSTRACT FROM AUTHOR]

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS. [ABSTRACT FROM AUTHOR]

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (μFFE) separation. Since μFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in μFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC X μFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC X μFFE peak capacities as high as 2.3S2 could be obtained in a 10 min separation window when determined according to die product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC X μFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC X LC. [ABSTRACT FROM AUTHOR]

While electrophoresis in lipid bilayers has been performed since the 1970s, the technique has until now been unable to accurately measure the charge on lipids and proteins within the membrane based on drift velocity measurements. Part of the problem is caused by the use of the Einstein–Smoluchowski equation to estimate the electrophoretic mobility of such species. The source of the error arises from the fact that a lipid headgroup is typically smaller than the Debye length of the adjacent aqueous solution in most electrophoresis experiments. Instead, the Henry equation can more accurately predict the electrophoretic mobility at sufficient ionic strength. This was done for three dye-labeled lipids with different sized head groups and a charge on each lipid of −1. Also, the charge was measured as a function of pH for two titratable lipids that were fluorescently labeled. Finally, it was shown that the Henry equation also has difficulties measuring the correct lipid charge at salt concentrations below 5 mM, where electroosmotic forces are more significant. [ABSTRACT FROM AUTHOR]

Better understanding of the mechanism is important for exploring the potentials of a preconcentration method. In this work, we show for the first time that the HF etched porous junction on a fused silica capillary behaves not only as a filter but also as an integrated nanofluidic interface. This junction exhibits an obvious ion concentration polarization (CP) effect, with which highly efficient electrokinetic stacking (ES) inside the capillary can be achieved without molecular size or charge type limitation. Two major types of CP based ES were proposed, and an autostop etching principle was presented for avoiding overetching. The ES can be performed in a broad range of pH and buffer concentration. Over a billion times of concentration was demonstrated by a fluorescein probe with laser induced fluorescent (LIF) detection. ES of fluorescently labeled and native DNA and protein were characterized by charge-coupled device (CCD) imaging and online capillary gel electrophoresis (CGE) with ultraviolet (UV) absorption detections, respectively. With this junction, highly efficient ES can be performed easily by voltage manipulation without any mechanical operation. We may foresee that the performance of capillary-based conventional and chip electrophoresis could be greatly enhanced with this junction in the analysis of low abundance biomolecules. [ABSTRACT FROM AUTHOR]

Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressure-driven flow prevent other sample components—for example, cells, proteins, or particulates in complex samples that can interfere with analysis—from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples. [ABSTRACT FROM AUTHOR]

Salts and buffers, commonly used in isolation and stabilization of biological analytes, have a deleterious effect on electrospray ionization mass spectrometry (ESI-MS). Excessive concentrations of salts lead to ion suppression and adduct formation, which mask or complicate ion signals. In this work, we describe a salt remover (SR), configured as a three-compartment flow-through system, where the central compartment is separated from the outer compartments by a cation-exchange membrane (CEM) and an anion-exchange membrane (AEM). One platinum electrode is placed in each of the outer compartments, where water or electrolyte is flowing. The CEM electrode is held at a negative potential relative to the AEM side; cations/anions migrate by electrophoresis to the CEM/AEM receiver liquids, respectively. Proteins have poorer electrophoretic mobility relative to the buffer components, permitting removal of the salt. The salt-free proteins proceed to the ESI source. The capillary scale SR (intemal volume 2.5 μL) described here effectively desalted continuous flows of NaCl solutions (200 mequiv/L at 1 μL/min, equivalent to a flux of 200 nequiv/min with 88% efficiency) and achieved >99.8% salt removal with 154 mM NaCl (isotonic saline) at 1 μL/min. With optimized current, >80% of concurrently present 20uM protein was transmitted. Desalting efficiency and analyte loss was evaluated with different salt concentration and flow rate combinations under different applied current. Good-quality ESI-MS spectra of cytochrome c, myoglobin, and lysozyine as test proteins in a saline solution, passed through the SR, are demonstrated. [ABSTRACT FROM AUTHOR]

The interplay between biophysical characteristics such as protein size and shape and protein function is difficult to ascertain using simple methods. Here, we present an approach for characterizing both protein—ligand binding as well as protein hydrodynamic radius in one operation combining electrophoresis and size measurement by dispersion using capillaries. The methodologr is based on the integration of Taylor dispersion analysis and capillary electrophoresis and is here demonstrated using commercially available capillaiy electrophoresis instrumentation modified with a pixel sensor UV area imager, allowing two detection points along the capillary. Analytes are the human serum proteins Ce1 -acid glycoprotein and albumin interacting with the drug propranolol in a frontal analysis mode. Upon introduction of the propranolol—protein sample, voltage is initially applied to facilitate electrophoretically mediated separation of ligand and protein and frontal analysis. Then a pressure mobilization step is used whereby Taylor dispersion can be characterized online based on the signal from the UV area imager. Estimates of ligand binding and values for hydrodynamic radii agree with values obtained by independent methods. [ABSTRACT FROM AUTHOR]

Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction-oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds. [ABSTRACT FROM AUTHOR]

N-Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes before the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. Technologies capable of characterizing eukaryotic and the newly described bacterial N-glycosylation systems from biologically relevant samples in a quick, accurate, and cost-effective manner are needed. In this paper a new glycomics strategy based on lectin-affinity capture was devised and validated on the C. jejuni N-glycan pathway and the engineered Escherichia coli strains expressing the functional C. jejuni pathway. The lipid-linked oligosaccharide intermediates of the Pgl pathway were then enriched using SBA-agarose affinity-capture and examined by capillary electrophoresis-mass spectrometry (CE-MS). We demonstrate that this method is capable of detecting low levels of LLOs, the sugars are indeed assembled on undecaprenylpyrophosphate, and structural information for expected and unexpected LLOs can be obtained without further sample manipulation. Furthermore, CE-MS analyses of C. jejuni and the E. coli "glyco-factories" showed striking differences in the assembly and control of N-glycan biosynthesis. [ABSTRACT FROM AUTHOR]

Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, rela- lively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10-20 mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations. [ABSTRACT FROM AUTHOR]

Attachment of a nonaggregating monoclonal antibody and of a soluble recombinant receptor molecule to the icosahedral nonenveloped human rhinovirus serotype 2 was studied with a nanoelectrospray ionization gas-phase electrophoretic molecular mobility analyzer (nESI-GEMMA). The virus mass, as determined via nESI-GEMMA, was within instrument accuracy (±6%) close to the theoretical value (8 x 106 Da) calculated from the sum of all constituents of one virus particle (60 copies of each of the four viral capsid proteins, the RNA genome, and one copy of the RNA-linked protein VpG). The formation of virus-antibody complexes of different stoichiometries (up to a mass 12.5 x 106 Da corresponding to 30 attached antibodies) and virus-receptor complexes (up to a mass 8.8 x 106 Da corresponding to 12 attached receptor molecules) was monitored. Via the volume derived from the electrophoretic mobility diameter (EMD), the stoichiometry of the HRV complexes was calculated. The accuracy of the EMD was within ±0.5 nm, which corresponds to an accuracy of ±4 antibodies and ±5 receptor molecules in the respective complexes. For the first time, we here demonstrate the use of nESI-GEMMA for the analysis of the size and stoichiometry of biomolecules in high-order complexes in real time under normal pressure conditions. [ABSTRACT FROM AUTHOR]

Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. [ABSTRACT FROM AUTHOR]

We report a dynamic cross-linking effect of Mg2+ that enhances the sieving properties of low-viscosity poly(vinylpyrrolidone) (PVP) solutions. A low-viscosity PVP solution was applied to nondenaturing microchip electrophoresis of protein samples using microchips made of poly(methyl methacrylate). The separation resolution of nondenatured protein markers in 1.8% PVP solution was improved by adding 1-20 mM MgC2. We studied the effect of the ratio of cross-linking agent on mobility of protein samples and showed that protein retardation (In μ/μ0) is correlated with the ratio of cross-linking agent to PVP ([cMg2+/cPVP]) as ln μ/μ0 = A′[cMg2+/cPVP]b′. A′ was related to the protein radius (R), and b′ was found to be 0.72 for proteins with R = 2.4 nm and 0.82 for proteins with R = 1.85 nm. A structural study of PVP in semidilute solutions using dynamic light scattering showed that incremental increases of Mg2+ ion concentration from 5 to 20 mM in 1.8% PVP solution increased the hydrodynamic radius of PVP polymers by 20%. [ABSTRACT FROM AUTHOR]

Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FVLE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201—213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface. [ABSTRACT FROM AUTHOR]

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates. [ABSTRACT FROM AUTHOR]

A robust, reproducible, and single-step interface design between low flow rate separation techniques, such as sheathless capillary electrophoresis (CE) and nanoliquid chromatography (nLC), and mass spectrometry (MS) using electrospray ionization (ESI), is introduced. In this design, the electrical connection to the capillary outlet was achieved through a porous tip at the capillary outlet. The porous section was created by removing 1–1.5 in. of the polyimide coating of the capillary and etching this section by 49% solution of HF until it is porous. The electrical connection to the capillary outlet is achieved simply by inserting the capillary outlet containing the porous tip into the existing ESI needle (metal sheath) and filling the needle with the background electrolyte. Redox reactions of water at the ESI needle and transport of these small ions through the porous tip into the capillary provides the electrical connection for the ESI and for the CE outlet electrode. The etching process reduces the wall thickness of the etched section, including the tip of the capillary, to 5–10 μm, which for a 20–30 μm i.d. capillary results in stable electrospray at 41.5 kV. The design is suitable for interfacing a wide range of capillary sizes with a wide range of flow rates to MS via ESI, but it is especially useful for interfacing narrow (<30 μm i.d.) capillaries and low flow rates (<100 nL/min). The advantages of the porous tip design include the following: (1) its fabrication is reproducible, can be automated, and does not require any mechanical tools. (2) The etching process reduces the tip outer diameter and makes the capillary porous in one step. (3) The interface can be used for both nLC-MS and CE-MS. (4) If blocked or damaged, a small section of the tip can be etched off without any loss of performance. (5) The interface design leaves the capillary inner wall intact and, therefore, does not add any dead volume to the CE-MS or nLC-MS interface. (6) Bubble formation due to redox reactions of water at the high-voltage electrode is outside of the separation capillary and does not affect separation or MS performances. The performance of this interface is demonstrated by the analyses of amino acids, peptide, and protein mixtures. [ABSTRACT FROM AUTHOR]

We have developed a simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method for trace analysis of DNA. The microchip comprised of three parallel channels: the first two are for the field-amplified sample stacking and subsequent field-amplified sampled injection steps, while the third one is for the microchip gel electrophoresis (MGE) with ED (MGE-ED). To improve preconcentration and separation performances of the method, the stacking and separation buffers containing the hydroxypropyl cellulose (HPC) matrix were modified with gold nanoparticles (AuNPs). The formation of AuNPs and HPC/AuNP-modified buffers were characterized by UV-visible spectroscopy and TEM experiments. The conducting polymer-modified electrode was also modified with AuNPs to enhance detection performances of the electrode. The conducting polymer/AuNP layers act as electrocatalysts for the direct detection of DNA based on their oxidation in a solution phase. The total sensitivity was improved by ∼25 000-fold when compared with a conventional MGE-ED analysis. The calibration plots were linear (r2 = 0.9993) within the range of 0.003-1.0 pg/μL for a 20-bp DNA sample. The sensitivity was 0.20 nA/(fg/μL), with a detection limit of 5.7 amol in a 50-μL sample, based on S/N = 3. The applicability of the method for the analysis of 13 fragments present in a 100-bp DNA ladder was successfully demonstrated. [ABSTRACT FROM AUTHOR]

A poly(ethylene glycol)-functionalized acrylic copolymer was developed for fabrication of microfluidic devices that are resistant to protein and peptide adsorption. Planar microcapillary electrophoresis (µCE) devices were fabricated from this copolymer with the typical cross pattern to facilitate sample introduction. In contrast to most methods used to fabricate polymeric microchips, the photopolyinerization-based method used with the copolymer reported in this work was of the soft lithography type, and both patterning and bonding could be completed within 10 min. In a finished microdevice, the cover plate and patterned substrate were bonded together through strong covalent bonds. Additionally, because of the resistance of the copolymer to adsorption, fabricated microfluidic devices could be used without surface modification to separate proteins and peptides. Separations of fluorescein isothiocyanate-labeled protein and peptide samples were accomplished using these new polymeric µCE microchips. Separation efficiencies as high as 4.7 ×104 plates were obtained in less than 40 s with a 3.5-cm separation channel, yielding peptide and protein peaks that were symmetrical. [ABSTRACT FROM AUTHOR]

High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confinned by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins: Src, SH2-Bβ, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-Bβ and Fyn complex barely affected. IC50 of both selective and nonselective inhibitors were determined and compared for different proteins. The IC50 of the nonselective inhibitor was 49 ± 9, 323 ± 42, and 228 ± 19 μM (n = 3) for Src, SH2-Bβ, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein-protein interactions. [ABSTRACT FROM AUTHOR]

Electrocapture is a multifunctional microfluidic tool that can be used for concentration, sample cleanup, multistep reactions, and separation of biomolecules. Herein, we investigate the mechanisms underlying the electrocapture principle. A microfluidic electrocapture device was found to be capable of generating regions of different electric field, which are maintained in the flow by electric and hydrodynamic forces, with the zones of lower electric field strength upstream of those with higher strength. In addition to detection of the local electric fields by direct measurements, the existence of the zones was observed by the capture of a solution containing Coomassie and myoglobin. The two molecules were captured at different spots in a steady-state manner and were released (separated) at different electric fields. Considering these observations and the experimental values for the electric field strengths, flow velocities, and electrophoretic mobilities of DNA, proteins, and peptides, it is concluded that the macromolecules are captured between the field zones by a stacking mechanism. [ABSTRACT FROM AUTHOR]

We present the first example of the analysis of acidic and basic proteins by nanoparticle-filled capillary electro-phoresis. Compared to the didodecyldimethylammonium bromide (DDAB)-coated capillary, the DDAB-capped gold nanoparticles (AuNPs) as pseudostationary phase were found to form more stable coating on the capillary wall, thus leading to greater separation efficiency and high reproducibility. In addition to their advantages for protein separation, DDAB-capped AuNPs can generate high reversed elecfroosmotic flow, which is 75% greater than DDAB at pH 3.5. To allow strong interactions with proteins, the AuNPs were modified with poly(ethylene oxide) via noncovalent bonding to form gold nanopar- tides/polymer composites (AuNPPs). Using a capillary dynamically coated with DDAB-capped AuNPs and filled with AuNPPs under acidic conditions (10 mM phosphate, pH 3.5), we have demonstrated the separation of acidic and basic proteins with peak efficiencies ranging from 71 000 to 1 007 000 plates/m and relative standard deviations of migration time less than 0.6%. Additionally, the proposed method has been applied to the analyses of biological samples, including saliva, red blood cells, and plasma. With simplicity, high resolving power, and high reproducibility, the proposed method has shown great potential for proteomics applications and clinical diagnosis. [ABSTRACT FROM AUTHOR]

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, Kd, of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (~44 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents. [ABSTRACT FROM AUTHOR]

We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an Ar+ laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1 000 000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes. [ABSTRACT FROM AUTHOR]

Efficient sample preparation tools are the key to measuring molecular signals in a complex biological system. A novel continuous-flow isoelectric point (pI)-based sorting technique has been developed for proteins and peptides in a microfluidic chip format. It can sort biomolecules at a relatively high flow rate of up to 10 µl/min and does not require carrier ampholytes, which can create molecular backgrounds for subsequent analysis. Furthermore, the electrophoretic field required to run the pI-based sorting is generated by the diffusion of buffer ions in situ, at the liquid junction between two laminar flows within the microfluidic channel. Utilizing the diffusion potential in combination with a pH difference between the buffers, we demonstrated a separation of binary mixtures of pI markers and proteins without applying any external field. The sorting resolution and its efficiency are sufficiently high for sample preparation and could be further improved by optimizing buffers or with an additional transverse electric field. Once fully developed, it can potentially be a pI-based sample fractionation tool for proteomic analysis of complex biomolecule samples. [ABSTRACT FROM AUTHOR]

Proteins visualized by 2,2,2-trichioroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time- of-flight (MALDI-TOF) mass spectrometry (MS) and MS/ MS. In a previous study, a method was developed that placed ICE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 mm. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of ICE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, ICE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. ICE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, ICE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, ICE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels. [ABSTRACT FROM AUTHOR]

Monitoring food quality is a critical task for analytical chemistry and an important way to preserve human health. Fish is a valuable source of highly digestible proteins and contains large amounts of polyunsaturated fatty acids and fat-soluble vitamins. Since the world's wild fish stocks are limited, farmed fish is nowadays proposed as an alternative to consumers. It is now emerging that the fish muscle protein content is assuming teat importance from an aquaculture perspective. Many data have been collected on the physiology and biochemistry of fish muscle, but few proteomic studies are available on farmed fish. Application of proteomics to aquaculture may play a key role in the development of new farming strategies. In this paper, a proteomic approach based on SDS-PAGE separation of proteins, in situ protein hydrolysis, de novo sequencing of peptides by MALDI and ESI MS², protein identification and relative quantitation of protein by denaturing capillary electrophoresis was coupled with the determination of fatty acids and metal ions content by GM-MS and ICPMS in farmed and wild sea bass filet Our results show that aquaculture could induce significant chemical and biochemical differences in fish muscle that may have an impact on food quality. [ABSTRACT FROM AUTHOR]