Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips

Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

High-speed separation of proteins by sodium dodecyl sulfate-capillary gel electrophoresis with partial translational spontaneous sample injection

In this study, we developed a picoliter-scale partial translational spontaneous injection approach which is suitable for high-speed protein separation under sodium dodecyl sulfate-capillary gel electrophoresis mode. On the basis of this approach, we built a high-speed CE system for protein separation based on a short capillary and slotted-vial array. The system has the advantages of simple structure, ease of building without the requirement of microfabricated devices, convenient operation, and low cost. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ～65?μm plug length) were obtained, which ensured both the high speed and the high efficiency in protein separation. Five fluorescein isothiocyanate labeled proteins including myoglobin, egg albumin, bovine serum albumin, phosphorylase b, and myosin were separated within 60?s with an effective separation length of 1.5?cm. Theoretical plates per meter ranging from 2.58×10? to 1.28×10? (corresponding to 0.78-3.88?μm plate height) were obtained. The separation speed and separation efficiency of the present system are comparable to those of most microchip-based capillary electrophoresis systems for protein separation. The relative standard deviations of the migration times were in the range of 0.9-1.3% (n=5). Good linear relationships between log relative molecular mass and migration time were obtained in the molecular weigh range of 17,200-500,000, which demonstrate the present system can be applied in protein relative molecular mass determination.     

On-line isotachophoretic preconcentration and gel electrophoretic separation of sodium dodecyl sulfate-proteins on a microchip

A microchip for integrated isotachophoretic (ITP) preconcentration with gel electrophoretic (GE) separation to decrease the detectable concentration of sodium dodecyl sulfate (SDS)-proteins was developed. Each channel of the chip was designed with a long sample injection channel to increase the sample loading and allow stacking the sample into a narrow zone using discontinuous ITP buffers. The pre-concentrated sample was separated in GE mode in sieving polymer solutions. All the analysis steps including injection, preconcentration, and separation of the ITP-GE process were performed continuously, controlled by a high-voltage power source with sequential voltage switching between the analysis steps. Without deteriorating the peak resolution, four SDS-protein analyses with integrated ITP-GE system resulted in a decreased detectable concentration of approximately 40-fold compared to the GE mode only. A good calibration curve for molecular weights of SDS-proteins indicated that the integrated ITP-GE system can be used for qualitative analysis of unknown protein samples.     

Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).     

Rapid and efficient isotachophoretic preconcentration in free solution coupled with gel electrophoresis separation on a microchip using a negative pressure sampling technique

We present a novel isotachophoresis–gel electrophoresis (ITP–GE) microchip system designed for rapid and efficient isotachophoretic preconcentration coupled with gel electrophoresis separation by using a negative pressure sampling technique. The overall ITP–GE procedure involves only three steps: sample loading, ITP preconcentration and GE separation and was controlled by a simple and compact negative pressure sampling device, which is composed of a vacuum vessel, a three-way electromagnetic valve and a single high voltage power supply. During the sample loading stage, a negative pressure was applied via a three-way electromagnetic valve in headspace of the two sealed sample waste reservoirs (SWs). A sandwiched sample zone between a leading and a terminating electrolyte zone was formed in the channel intersection in less than 1 s. Once the three-way electromagnetic valve was switched to connect SWs to ambient atmosphere to release vacuum in SWs, ITP preconcentration in free solution and GE separation in the 4% hydroxyethylcellulose (HEC) sieving material were consequently activated under the electric potentials applied. The performance of present approach was evaluated by using DNA fragments as model analytes. Compared to conventional cross microchip GE using electrokinetic pinched injection, an average signal enhancement of 185-fold was obtained with satisfactory resolution. The results demonstrated the ITP–GE approach possessing an exciting potential of high sensitivity and short sampling time with significant simplification in operation and instrumentation.     

Brush-like copolymer as a physically adsorbed coating for protein separation by capillary electrophoresis

A brush-like copolymer consisting of poly(ethylene glycol) methyl ether methacrylate and N,N-dimethylacrylamide (PEGMA-DMA) was synthesized and used as a novel static physically adsorbed coating for protein separation by capillary electrophoresis for the first time, in order to stabilize electroosmotic flow (EOF) and suppress adsorption of proteins onto the capillary wall. Very stable and low EOF was obtained in PEGMA-DMA-coated capillary at pH 2.2-7.8. The effects of molar ratio of PEGMA to DMA, copolymer molecular mass, and pH on the separation of basic proteins were discussed. A comparative study of bare capillary with PEGMA-DMA-coated capillary for protein separation was also performed. The basic proteins could be well separated in PEGMA-DMA-coated capillary over the investigated pH range of 2.8-6.8 with good repeatability and high separation efficiency because the copolymer coating combines good protein-resistant property of PEG side chains with excellent coating ability of PDMA-contained backbone. Finally, the coating was successfully applied to the fast separation of other protein samples, such as protein mixture and egg white, which reveals that it is a potential coating for further proteomics analysis.     

Emulsion copolymerization of styrene and methacrylic acid in the presence of a polyethylene oxide based-polymerizable stabilizer with a shorter chain length

The emulsion copolymerization of styrene and methacrylic acid (MAA) was performed in the presence of a relatively new macromonomer, poly(ethylene glycol) ethyl ether methacrylate (PEG-EEM) as a stabilizer. In contrast to similar studies, a macromonomer having relatively shorter polyethylene oxide chain length (i.e., M_n:246, n ≈ 3.0) was selected for this study. Highly uniform and carboxyl functionalized latex particles in the size range of 0.16–0.50 μm were obtained by changing MAA, PEG-EEM, total monomer, and initiator concentrations. The use of PEG-EEM as a stabilizer resulted in larger monodisperse particles relative to those obtained by the emulsifier-free emulsion copolymerization of styrene and MAA. The particle size decreased and the polymerization rate increased with the increasing MAA feed concentration. The application of power law model indicated that MAA concentration was more effective in the presence of PEG-EEM for control of particle size relative to similar systems. The latex particles with higher numbers of surface-carboxyl groups were obtained with the higher MAA feed concentrations. Although the particle size decreased and the polymerization rate increased with the increasing PEG-EEM concentration in the emulsion polymerization of styrene, both of them remained roughly constant with the increasing PEG-EEM concentration in the presence of MAA. Received: 21 December 2000 Accepted: 13 July 2000     

Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis

To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis.     

Size-exclusion separation of proteins using a biocompatible polymeric monolithic capillary column with mesoporosity

Biocompatible poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared for size exclusion chromatography (SEC) of proteins in the capillary format using Brij 58P in a mixture of hexanes and dodecanol as porogens. The monolithic columns provided size separation of four proteins in 20 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights. Compared to SEC monoliths previously synthesized using a triblock copolymer of polyethylene oxide and polypropylene oxide, an increase in mesoporosity was confirmed by inverse size exclusion chromatography. As a result, improved protein separation in the high molecular weight range and reduced column back-pressure were observed.     

Improved detection of human pancreatic proteins separated by two-dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis using a combined staining procedure.

In order to assess secretory pancreatic proteins in a two-dimensional isoelectric focusing/sodium dodecyl sulphate electrophoresis gel, a highly sensitive double-staining method with Coomassie Brilliant Blue followed by silver stain was used. This combined procedure afforded more distinct spots and additional bands, particularly glycoproteins, than either silver or Coomassie Blue staining alone. As measurements of dye volumes by densitometry have shown, double staining of two-dimensional separated pancreatic proteins is up to twenty times more sensitive than the usual Coomassie Brilliant Blue staining.     

Analysis of a biopolymer by capillary electrophoresis with a chemiluminescence detector using a polymer solution as the separation medium.

We developed capillary electrophoresis with a chemiluminescence detector using a polymer solution as the separation medium for the analysis of biopolymers, such as DNA and protein. A peroxyoxalate chemiluminescence reagent of bis(2,4,6-trichlorophenyl)oxalate was used together with fluorescein-labeling reagent. When a migration buffer solution containing carboxylmethylcellulose was used, the flow-type chemiluminescence detection cell was found to give a better resolution than the batch-type one. Fluorescein-labeled adenosine triphosphate of 1.0 x 10(-4) M was examined by means of capillary electrophoresis with absorption (260 nm), fluorescence (ex. 496 nm and em. 517 nm), and chemiluminescence detectors. The chemiluminescence detection showed the highest sensitivity among them; the S/N ratios obtained by absorption, fluorescence, and chemiluminescence detections were 4, 38, and 130, respectively. Fluorescein-labeled DNA was prepared through a polymerase chain reaction using fluorescein-labeled deoxyadenosine triphosphate. A mixture of the labeled DNA fragments (500, 600, 700, 800, 900, and 993 bp) was successfully separated and detected by the present system. A mixture of proteins (lysozyme, cytochrome C, and ribonuclease A) which were labeled with fluorescein isothiocyanate was also separated and detected.     

Size-based separations of proteins by capillary electrophoresis using linear polyacrylamide as a sieving medium: model studies and analysis of cider proteins

Electrophoretic conditions to separate sodium dodecyl sulfate (SDS)-protein complexes according to their relative molecular mass by capillary electrophoresis (CE) using linear polyacrylamide as a sieving matrix were examined. Five purified proteins with relative molecular masses between 14 400 and 66 200 Da were separated on a coated fused-silica capillary with an internal diameter of 100 microm and an effective length of 24 cm (total length, 32.5 cm). Benzoic acid was added to the solution of purified proteins as internal standard; beta-mercaptoethanol was also added as reducing agent. The running buffer composition was 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.035 M aspartic acid, 0.1% m/v SDS, 4% m/v acrylamide, the resulting pH being 8.0. The applied voltage was 7 kV (reversed voltage polarity) in order to avoid high current intensities. Under optimized conditions, the five proteins were separated in less than 15 min, with a % relative standard deviation (RSD) between 0.2 and 0.4 for migration times in the same day. Good efficiency (values between 150 000 and 40 000 N/m) and resolution (values between 2 and 2.8) were obtained. The inverse of relative migration times was found to correlate with the logarithm of their relative molecular mass. Finally, cider proteins were analyzed and their relative molecular masses were determined. These results were compared with those obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).     

毛细管筛分电泳中电动超荷电结合预填充水塞在线富集十二烷基磺酸钠-蛋白质的复合物

提出了一种应用于毛细管筛分电泳中的电动超荷电结合柱头水塞堆积样品的方法,实现了十二烷基磺酸钠-蛋白质复合物的在线富集。一般情况下,电动超荷电方法是一种将电动进样与瞬时等速电泳联用的富集技术。具体过程是,首先在毛细管中注入背景电解质,再注入适量的前导电解质,然后电动进样一段时间。最后注入后导电解质开始瞬时等速电泳及分离的过程。本文在常规的电动超荷电技术基础上,在电动进样之前先注入一段含有聚合物的水塞以进一步提高富集效果。同时,考察了电动超荷电中不同富集方法叠加联用的效果,包括聚合物的筛分效应、结合水塞和不结合水塞的场放大样品进样效果、瞬时等速电泳等。结果表明,由于十二烷基磺酸钠-蛋白质复合物的质荷比接近,电动进样中的进样歧视得到消除,电动超荷电结合含聚合物水塞堆积样品的方法可以无歧视地在线富集十二烷基磺酸钠-蛋白质复合物,检测灵敏度增强1000倍以上。该方法非常适用于低丰度蛋白质的分析,并可同时提供相对分子质量信息。     

Identification of proteins separated by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis with matrix-assisted laser desorption/ionization ion trap mass spectrometry; comparison with matrix-assisted laser desorption/ionization time-of-flight mass fingerprinting

Digests from ten gel bands containing low abundance proteins were analyzed by both matrix-assisted laser desorption/ionization ion trap (MALDI-IT) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) methods. MALDI-TOF techniques were able to identify only one protein from all 10 gel bands, while MALDI-IT identified eight proteins from the same 10 bands. The ability to perform MS/MS experiments with a MALDI-IT instrument leads to protein identifications based on both peptide molecular mass and sequence information, and is much less prone to errors and uncertainties introduced by peptide fingerprinting methodologies in which protein identification is based on peptide molecular masses alone.     

One-pot synthesis of aryl alkyl thioethers and diaryl disulfides using carbon disulfide as a sulfur surrogate in the presence of diethylamine catalyzed by copper(I) iodide in polyethylene glycol (PEG200)

A copper catalyzed one-pot protocol for the preparation of aryl alkyl thioethers and diaryl disulfides using carbon disulfide as the sulfur source and diethylamine in polyethylene glycol (PEG200) is described.     

Assembly of poly(dopamine)/poly(acrylamide) mixed coatings by a single‐step surface modification strategy and its application to the separation of proteins using capillary electrophoresis

In this work, a facile approach was developed to modify a fused‐silica capillary inner surface based on poly(dopamine) and poly(acrylamide) mixed coatings for protein separation by capillary electrophoresis. The surface morphology, thickness, and chemical components of poly(dopamine)/poly(acrylamide) mixed coatings on glass slides and silicon wafers were studied by atom force microscopy, ellipsometry, and X‐ray photoelectron spectroscopy, respectively. The hydrophilicity and stability of the mixed coatings on glass slides were investigated by static water contact angle measurements. A comparative study of electroosmotic flow showed that the poly(dopamine)/poly(acrylamide) mixed coatings could provide effective suppression of electroosmotic flow. Meanwhile, the fast and efficient separations of the mixture of four alkaline proteins, the mixture of acidic, basic, and neutral proteins and egg white proteins were obtained by capillary electrophoresis. Furthermore, the consecutive protein separation runs and low RSDs of migration time demonstrated that these poly(dopamine)/poly(acrylamide) mixed coatings were capable of minimizing protein adsorption during the protein separation by using capillary electrophoresis.     

Preparation of nanocrystalline porous titania films on titanium substrates by a sol–gel method with polyethylene glycol as a template

Porous nanocrystalline TiO₂ films have been prepared on cp-Ti substrates for biomedical usage by a sol–gel process from the solutions containing polyethylene glycol (PEG) as a template. Variations of the crystal structure with heat-treatment temperature determined by XRD are different for TiO₂ films and powders, due to the effect of titanium substrate. The surface texture of porous TiO₂ films is analyzed by means of SEM and found to greatly depend on the concentration and molecular weight of PEG. The pore formation mechanism is discussed in relation to the self-assembly of PEG and phase separation between PEG adsorbed on TiO₂ oligomers and ethanol.     

Synthesis of tetracycline‐imprinted polymer microspheres by reversible addition–fragmentation chain‐transfer precipitation polymerization using polyethylene glycol as a coporogen

Tetracycline (TC)‐imprinted microspheres have been synthesized by reversible addition–fragmentation chain‐transfer precipitation polymerization using PEG as a coporogen. In the synthesis, methacrylic acid and ethylene dimethacrylate were used as the functional monomer and cross‐linker, respectively. 2,2′‐Azobisisobutyronitrile was the initiator, and cumyl dithiobenzoate was the chain‐transfer reagent. Although monodispersed microspheres were obtained using acetonitrile as porogen, the particles cannot be used in the column extraction because of the high backpressure. To increase the porosity of the material, PEG was introduced as a coporogen. The influence of the molecular weight and concentration of PEG on the morphology, binding affinity, and porosity of the molecularly imprinted polymers (MIPs) have been studied. The results demonstrated that PEG as a macroporogen increased the porosity of the polymers. Meanwhile, the column backpressure was reduced using the MIPs with higher porosity. The binding affinity of the MIPs was increased when a low concentration of PEG was employed, while it was decreased when the ratio of PEG 12 000/monomers was >0.8%. Under the optimized conditions, TC‐imprinted microspheres with good selectivity and size uniformity have been obtained, which facilitates its application in the column extraction for TC determinations.     

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.