Orders-of-Magnitude Larger Force Demonstrated for Dielectrophoresis of Proteins Enabling High-Resolution Separations Based on New Mechanisms.

Proteins are perhaps the most important yet frustratingly complicated and difficult class of compounds to analyze, manipulate, and use. One very attractive option to characterize and differentially concentrate proteins is dielectrophoresis, but according to accepted theory, the force on smaller particles the size of proteins is too low to overcome diffusive action. Here, three model proteins, immunoglobulin G, α-chymotrypsinogen A, and lysozyme, are shown to generate forces much larger than predicted by established theory are more consistent with new theoretical constructs, which include the dipole moment and interfacial polarizability. The forces exerted on the proteins are quantitatively measured against well-established electrophoretic and diffusive processes and differ for each. These forces are orders of magnitude larger than previously predicted and enable the selective isolation and concentration of proteins consistent with an extremely high-resolution separation and concentration system based on the higher-order electric properties. The separations occur over a small footprint, happen quickly, and can be made in series or parallel (and in any order) on simple devices.

Delayed Desorption Improves Protein Analysis by Desorption Electrospray Ionization Mass Spectrometry.

Protein analysis by desorption electrospray ionization mass spectrometry (DESI-MS) is limited and often accompanied by a mass-dependent loss in sensitivity as protein molecular weight increases. Previously, incomplete dissolution was identified as a potential contributing factor to this limitation for larger proteins. Here, we developed a unique two-step configuration in which a prewetting solvent is applied to the sample surface proximal to DESI analysis by a wetting quill to increase dissolution time and the detection of larger proteins. After optimizing the system with a mixture of proteins containing cytochrome c, myoglobin, and chymotripsinogen, we demonstrate the ability of delayed desorption to improve the analysis of larger proteins such as bovine serum albumin. Albumin and other serum proteins, including even larger ones, were also detected directly from diluted goat serum. An additional feature of this technique is the ability to deliver multiple solvents with potential synergistic or cooperative effects. For example, when using acetonitrile solutions of formic acid and ammonium bicarbonate as the prewetting and DESI spray solvent, respectively, the intensity of chymotrypsinogen improved dramatically compared to controls but less so for smaller proteins such as myoglobin and cytochrome c. Adduct removal was also observed for all proteins. These early results demonstrate the ability of this two-step technique for the use of multiple additives and increased dissolution times compared to standard DESI-MS experiments.

Online Hydrophobic Interaction Chromatography-Mass Spectrometry for Top-Down Proteomics.

Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.

Displacement chromatography of proteins using a retained pH front in a hydrophobic charge induction chromatography column.

The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.

Sensitive, high throughput detection of proteins in individual, surfactant-stabilized picoliter droplets using nanoelectrospray ionization mass spectrometry.

Droplet-based fluidics is emerging as a powerful platform for single cell analysis, directed evolution of enzymes, and high throughput screening studies. Due to the small amounts of compound compartmentalized in each droplet, detection has been primarily by fluorescence. To extend the range of experiments that can be carried out in droplets, we have developed the use of electrospray ionization mass spectrometry (ESI-MS) to measure femtomole quantities of proteins in individual pico- to nanoliter droplets. Surfactant-stabilized droplets containing analyte were produced in a flow-focusing droplet generation microfluidic device using fluorocarbon oil as the continuous phase. The droplets were collected off-chip for storage and reinjected into microfluidic devices prior to spraying the emulsion into an ESI mass spectrometer. Crucially, high quality mass spectra of individual droplets were obtained from emulsions containing a mixture of droplets at >150 per minute, opening up new routes to high throughput screening studies.

Rapid and highly sensitive protein extraction via cobalt oxide nanoparticle-based liquid-liquid microextraction coupled with MALDI mass spectrometry.

A new approach for rapid and highly sensitive protein extraction using cobalt oxide nanoparticles modified with cetyltrimethylammonium (Co(3)O(4)/CTA(+) NP) using nanoparticle-based liquid-liquid microextraction (NP-LLME) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully demonstrated. For the first time, the metal oxide NPs (Co(3)O(4)/CTA(+) NP) prepared in the organic phase (toluene) have been successfully applied for the extraction and preconcentration of proteins from sample solutions and complex samples via electrostatic forces involved between the metal oxide NP and proteins. Lysozyme was used as the model protein to investigate the optimal extraction parameters of the current approach. The optimal conditions were obtained at pH > pI for 10 min of incubation time (extraction time) with 3% salt (NaCl) addition. The Co(3)O(4)/CTA(+) NP was successfully applied for the highly sensitive analysis of an array proteins such as insulin, chymotrypsinogen and lysozyme from aqueous solution, protein mixture and milk samples in nanoparticle-based liquid-phase microextraction coupled with MALDI-MS. The potentiality of the NP-LLME using Co(3)O(4)/CTA(+) NP for the extraction of proteins was also compared with other types of NP-liquid phase microextraction (LPME) methods. The current approach offers distinct advantages including rapidity, straightforwardness, high sensitivity for washing- and separation-free MALDI-MS analysis of proteins.

Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis.

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.

Single cell analysis in full body quartz glass chips with native UV laser-induced fluorescence detection.

In order to investigate the individual and inhomogenous cellular response, e.g. to external stimuli, single cell analysis is mandatory and may provide new cognitions in proteomics as well as in other fields of systems biology in the future. Here, we report on novel chip architectures for single cell analysis based on full body quartz glass microfluidic chips (QG chips) that extend our previous studies in polydimethylsiloxane (PDMS) chips, and enhance the detection sensitivity of native UV laser-induced fluorescence (UV-LIF) detection. Detection of a 10nM tryptophan solution with an S/N ratio of 11.9, which gives a theoretical limit of detection of 2.5 nM (with S/N=3), was possible. With these optimizations the three proteins alpha-chymotrypsinogen A, ovalbumin and catalase each at a concentration of 100 microg/mL (equal to 4 microM, 0.4 microM and 2.2 microM) were injected electrokinetically and could be separated with nearly baseline resolution. Furthermore, fluorescence spectra (excitation wavelength, lambda(ex) = 266 nm) clearly demonstrate the favourable properties like the very high UV transparency and the nearly vanishing background fluorescence of the QG chips as compared to PDMS chips and to PDMS quartz window (PQW) chips. Finally we exploit the improved sensitivity for single cell electropherograms of Spodoptera frugiperda (Sf9) insect cells.

Glomerular charge barrier and development of proteinuria in passive Heymann nephritis.

BACKGROUND/AIM: Passive Heymann nephritis (PHN) in rats is a commonly used model of membranous glomerulonephritis in man where the cause of proteinuria is not fully resolved. This study was designed to investigate the role of the glomerular charge barrier in development of PHN proteinuria. METHODS: We studied female Sprague-Dawley rats (n = 33) at days 0, 2, 5 and 14 after induction of PHN by injection of antiserum against renal tubular epithelial antigens (anti-Fx1A). Measuring clearance of chymotrypsinogen A (Chym, MW 25,000, 21 A) and similar sized anionic chymotrypsinogen (aChym), together with (51)Cr-EDTA, we hypothesized an increased sieving coefficient (theta) of aChym in the early phase of PHN due to impairment of the glomerular charge barrier. RESULTS: No proteinuria was seen at day 2 (5.8 +/- 1.4 mg/ 24 h, p > 0.05), while protein excretion increased to 23.2 +/- 2.9 mg/24 h (p < 0.05) at day 5 (84 +/- 2% albumin) and further to 544.3 +/- 51.1 mg/24 h (p < 0.01) at day 14 (60 +/- 1% albumin; p < 0.01, day 5 vs. day 14). theta aChym was similar to control at day 2 (0.49 +/- 0.03, p > 0.05), increased at day 5 to 0.62 +/- 0.02 (p < 0.01) but decreased at day 14 to 0.39 +/- 0.02 (p < 0.01). The sieving coefficient of Chym (theta Chym) was decreased at day 14 (p < 0.05). The ratio of theta aChym to theta Chym was increased at day 5 (p < 0.01) but was elsewhere similar to control. CONCLUSION: The increased ratio of theta aChym to theta Chym and selective albuminuria at day 5 indicates an initial impairment of the glomerular charge barrier in PHN.

Desorption electrospray ionization-mass spectrometry of proteins.

Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the detection of proteins ranging in molecular mass from 12 to 66 kDa. Proteins were uniformly deposited on a solid surface without pretreatment and analyzed with a DESI source coupled to a quadrupole ion trap mass spectrometer. DESI-MS parameters optimized for protein detection included solvent flow rate, temperature of heated capillary tube, incident and reflection angle, sheath gas pressure, and ESI voltage. Detection limits were obtained for all protein standards, and they were found to decrease with decreasing protein molecular mass: for cytochrome c (12.3 kDa) and lysozyme (14.3 kDa) a detection limit of 4 ng/mm2 was obtained; for apomyoglobin (16.9 kDa) 20 ng/mm2; for beta-lactoglobulin B (18.2 kDa) 50 ng/mm2; and for chymotrypsinogen A (25.6 kDa) 100 ng/mm2. The DESI-MS analysis of higher molecular mass proteins such as ovalbumin (44.4 kDa) and bovine serum albumin (66.4 kDa) yielded mass spectra of low signal-to-noise ratio, making their detection and molecular weight determination difficult. In this study, DESI-MS proved to be a rapid and robust method for accurate MW determination for proteins up to 17 kDa under ambient conditions. Finally, we demonstrated the DESI-MS detection of the bacteriophage MS2 capsid protein from crude samples with minimal sample preparation.

Rapid and easy protein staining for SDS-PAGE using an intramolecular charge transfer-based fluorescent reagent.

High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.

Sub-2 microm porous and nonporous particles for fast separation in reversed-phase high performance liquid chromatography.

One approach to achieve fast and efficient separations in packed column liquid chromatography (LC) is to reduce eddy diffusion and mass transfer resistance in the mobile phase using short columns packed with small particles. In this study, efficiencies of columns packed with 1.5 and 3.0 microm nonporous and porous particles were compared in reversed-phase LC using nitromethane and a protein as analytes. Nonporous particles provided overall higher efficiency at high linear velocities when nitromethane was used as solute; however, the efficiency difference diminished significantly when the particle size was reduced from 3 to 1.5 microm. Efficiencies for 1.5 microm nonporous particles were considerably higher than those for 1.5 microm porous particles at high linear velocities when a protein, alpha-chymotrypsinogen A (MW 25,000), was used as solute. In addition, the average retention factor for amylbenzene in a column packed with ACQUITY C(18) porous particles was approximately 16 fold higher than for Micra C(18) nonporous particles for aqueous mobile phase compositions containing from 40 to 75% acetonitrile. Pressure drop, sample loading capacity, and separation power were also evaluated and compared for porous and nonporous particles under practical conditions.

Evaluation and comparison of alternatives to Protein A chromatography Mimetic and hydrophobic charge induction chromatographic stationary phases.

In this paper Protein A mimetic and hydrophobic charge induction chromatographic (HCIC) stationary phases are characterized in terms of their protein adsorption characteristics and their selectivity is compared with Protein A chromatography using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were employed to compare the selectivities of these resins for both non-IgG model proteins as well as antibodies and the fusion proteins. While none of the non-IgG model proteins were observed to bind to the Protein A resin, most of them did in fact bind to the alternative resins. In addition, while the elution pH was similar for the model proteins and antibodies on the HCIC resin, the mimetic resins did exhibit higher binding for the antibodies under these linear pH gradient conditions. A mixed mode preparative isotherm model previously developed for HCIC was shown to accurately describe the adsorption behavior of the mimetic materials as well. Host cell protein clearance profiles were also investigated under preparative conditions using complex biological feeds and the results indicated that while some selectivity was observed for both the HCIC and the mimetic materials, the purification factors were in general significantly less than those obtained with Protein A. It is important to note, however, that the selectivity of the mimetic and HCIC materials was also observed to be antibody specific indicating that further optimization may well result in increased selectivities for these materials.

Novel adsorptive polyamine coating for enhanced capillary electrophoresis of basic proteins and peptides.

In capillary electrophoresis (CE), the anionic and hydrophobic nature of the fused-silica capillary surface has long been known to present a problem in protein and peptide analysis. The use of capillary surface coating is one of the approaches to avoid the analyte-wall interactions. In this study, a new polymer, poly-LA 313, has been synthesized, physico-chemical characterized, and applied as polyamine coating for CE separations. The coating process is highly reproducible and provides fast separations of peptides and proteins in a few minutes and with high efficiency. The physically adsorbed polymer gives rise to a durable coating in the range of pH 2-10, in the presence of organic modifiers (acetonitrile and methanol) and with complex biological samples. The efficiency of the new cationic polymer was also tested performing protein and peptide separations with capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS).

Double-layer fluorescent zymography for processing protease detection.

We have developed a novel double-layer zymographic method for the detection of specific processing proteases of a target proprotease using a specific fluorescent substrate. The target processing proteases were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was subsequently incubated with the target proenzyme used as the substrate. A cellulose acetate membrane was immersed in 10% glycerol and then soaked in the fluorescent substrate solution. The slab gel of the processing protease was covered with the fluorescent substrate membrane, making a double layer. The double layer was incubated at 37 degrees C, and the released fluorescent band, in which the processing protease was located, was detected using UV light. The advantages of the double-layer fluorescent zymographic method are as follows: (i) the specific detection of target proprotease using a specific substrate, (ii) a relatively rapid and sensitive method, (iii) effective detection using small amounts of crude material, and (iv) wide applications that include the detection of processing proteases and activators for target proteases. Typical examples used for the detection of the processing proteases, such as plasminogen activator, chymotrypsinogen activator, procaspase-3 processing protease and caspase-3 activators, using this new method are described in this article.

Deep UV laser-induced fluorescence detection of unlabeled drugs and proteins in microchip electrophoresis.

Deep UV fluorescence detection at 266-nm excitation wavelength has been realized for sensitive detection in microchip electrophoresis. For this purpose, an epifluorescence setup was developed enabling the coupling of a deep UV laser into a commercial fluorescence microscope. Deep UV laser excitation utilizing a frequency quadrupled pulsed laser operating at 266 nm shows an impressive performance for native fluorescence detection of various compounds in fused-silica microfluidic devices. Aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan could be detected at low-micromolar concentrations. Deep UV fluorescence detection was also successfully employed for the detection of unlabeled basic proteins. For this purpose, fused-silica chips dynamically coated with hydroxypropylmethyl cellulose were employed to suppress analyte adsorption. Utilizing fused-silica chips permanently coated with poly(vinyl alcohol), it was also possible to separate and detect egg white chicken proteins. These data show that deep UV fluorescence detection significantly widens the application range of fluorescence detection in chip-based analysis techniques.

Direct measurement of protein osmotic second virial cross coefficients by cross-interaction chromatography.

The importance of weak protein interactions, such as protein self-association, is widely recognized in a variety of biological and technological processes. Although protein self-association has been studied extensively, much less attention has been devoted to weak protein cross-association, mainly due to the difficulties in measuring weak interactions between different proteins in solution. Here a framework is presented for quantifying the osmotic second virial cross coefficient directly using a modified form of self-interaction chromatography called cross-interaction chromatography. A theoretical relationship is developed between the virial cross coefficient and the chromatographic retention using statistical mechanics. Measurements of bovine serum albumin (BSA)/lysozyme cross-association using cross-interaction chromatography agree well with the few osmometry measurements available in the literature. Lysozyme/alpha-chymotrypsinogen interactions were also measured over a wide range of solution conditions, and some counterintuitive trends were observed that may provide new insight into the molecular origins of weak protein interactions. The virial cross coefficients presented in this work may also provide insight into separation processes that are influenced by protein cross-interactions, such as crystallization, precipitation, and ultrafiltration.

A hybrid model framework for the optimization of preparative chromatographic processes.

An optimization framework based on the use of hybrid models is presented for preparative chromatographic processes. The first step in the hybrid model strategy involves the experimental determination of the parameters of the physical model, which consists of the full general rate model coupled with the kinetic form of the steric mass action isotherm. These parameters are then used to carry out a set of simulations with the physical model to obtain data on the functional relationship between various objective functions and decision variables. The resulting data is then used to estimate the parameters for neural-network-based empirical models. These empirical models are developed in order to enable the exploration of a wide variety of different design scenarios without any additional computational requirements. The resulting empirical models are then used with a sequential quadratic programming optimization algorithm to maximize the objective function, production rate times yield (in the presence of solubility and purity constraints), for binary and tertiary model protein systems. The use of hybrid empirical models to represent complex preparative chromatographic systems significantly reduces the computational time required for simulation and optimization. In addition, it allows both multivariable optimization and rapid exploration of different scenarios for optimal design.

Mechanism of action of quaternary diamino quenchers in capillary zone electrophoresis.

The synthesis of two novel amino compounds, able to quench and reverse the electroosmotic low (EOF) in capillary electrophoresis is here reported. These chemicals are derivatives of two previously described quaternarized piperazines, 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4I) and 1-(4-iodobutyl)4-aza-1-azoniabicyclo[2.2.2] octane iodide (M7C4I), believed to bind covalently to the silica surface via alkylation of ionized silanols through their terminal, reactive iodine. The novel compounds, although unable to form a covalent bond, due to lack of the o-butyl iodine, are found to be very efficient in suppressing protein interaction with the wall and reversing the EOF. On the basis of their behavior in solution, the minimal structural motifs for strong binding of amino compounds to the silica wall have been derived and are thought to be: (i) the presence of two quaternary nitrogens in the molecule; (ii) a correct distance between said charged nitrogens, represented by a butyl chain (C(4)); (iii) an hydrophobic "decoration" of the molecules, consisting on a high CH(2)/N ratio (in the present case 8:1).