Capillary electrophoretic analysis of wild type and mutant Proteus penneri outer membrane proteins

In this study the virulence factors, outer membrane proteins (OMP), lipopolysaccharides (LPS), hemolysin, and the in vivo and in vitro virulence of wild-type Proteus penneri 357 and its two isogenic mutant variants--a transposon and a spontaneous mutant--were examined. The OMPs of these variants were analyzed by a new and fast technique, "dynamic sieving" capillary electrophoresis (CE). The OMP profiles were dominated by two peaks (39 and 43 kDa). In the P. penneri clone examined, both the transposon and the spontaneous mutations induced significant changes in the OMP patterns (in the relative percentage of the dominant proteins). CE was suitable for the comparative analysis of bacterial protein patterns in the genetic variants of this strain, and provided valuable results in connection with the bacteriological virulence. The LPS composition of the genetic variants also showed alterations. The wild type of P. penneri 357 showed a typical ladder pattern, an "S" form, and the mutants possessed "R" LPS patterns (only few bands) in the gels. In the bacteriological virulence tests the wild type of P. penneri 357 was virulent in the in vivo, and toxic in the in vitro assays, while both mutants showed neither toxicity nor pathogenicity.     

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%).     

微流体芯片电泳技术对人血清蛋白的快速分离

通过微流体芯片电泳技术分离人血清蛋白,探讨了常见十字形微流体芯片上样品的电动进样与分离过程,分析了在十字芯片上的进样时间和电压设置对后续样品检测和定量的影响。采用的缓冲体系为:100mmol/L H3BO3,50mmol/L NaCl,5%Dextran(以NaOH调至pH 8.3),该缓冲液能够有效分离人血清蛋白中的白蛋白(Albumin)和4种球蛋白(α1-,α2-,β-,和γ-globulin),并且给出了它们在该缓冲体系中的淌度估算范围为5.15×10-5～47.2×10-5 cm2/(V.s)。在芯片上2min之内可以完成进样和分离,相比于常用的毛细管区带电泳,提高了分析速度。     

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.     

毛细管电泳微芯片在临床尿蛋白检测中的应用研究

用微芯片毛细管电泳法对临床患者尿蛋白进行了分离, 初步探讨了用于判断肾损伤的应用前景. 以pH 10.3, 75 mmol•L^－1的硼酸盐缓冲液作为芯片电泳缓冲体系, 利用蛋白质的紫外吸收特性, 在210 nm波段检测吸光度并进行信号收集和分析. 研究两种添加剂对提高尿蛋白分离效率的影响, 分析了肾病综合症、妊娠高血压症、风湿性心脏病和多发性骨髓瘤等患者尿样本, 并与美国Helena电泳系统分析结果对比, 得到了较一致的结果.     

Lab‐on‐a‐Chip Biosensor for Glucose Based on a Packed Immobilized Enzyme Reactor

In this work, the development of a packed immobilized enzyme reactor (IMER) and its integration to a capillary electrophoresis microchip is described. The present microchip design differs from others, in the fact that the same design could be used with or without the particles and, just by changing the material used to pack the IMER, different analytes can be detected. The applied procedure involves the separation of the target analyte by capillary electrophoresis (CE), which is then coupled to a post‐column IMER that produces H₂O₂. The H₂O₂ produced is finally detected downstream at the surface of a working electrode. Glucose was detected above 100 μM by packing particles modified with glucose oxidase at the end of the separation channel. The analytical performance of the microchip‐CE has been demonstrated by performing the separation and detection of glucose and noradrenaline. Additions of fructose showed no effect on either the peak position or the peak magnitude of glucose. The microchip‐CE‐IMER was also used to quantify glucose in carbonated beverages with good agreement with other reports.     

Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35

Bacterial strains have complex and individual antigenic structure, which provides basis for their serological identification. However, serological cross-reaction may occur when antibodies against a certain strain recognize other strains too. The molecular basis of this phenomenon is the expression of similar or identical antigenic epitopes on the surface of different bacterial cells. Such cross-reactions might harden the serological diagnosis of pathogenic bacteria. But it can be also advantageous, when antigens of non-pathogenic strains can be used in the serological examinations. Serological cross-reaction between three taxonomically different strains--Proteus morganii O34 (8662/64), Escherichia coli O111 and Salmonella Adelaide O35--have been described. It has been proven that it is based partially on the similar lipopolysaccharide structures of these pathogens. In this study the involvement of the outer membrane proteins of these strains in the serological cross-reaction is presented. Microfluidic chip technology was applied for the detection of common proteins, which provided fast and quantitative data about the proteins that might be responsible for serological cross-reaction. Two outer membrane proteins with apparent molecular mass of 36 and 41 kDa, respectively, could be detected in the profile of each strain, while individual dominating protein peaks have also appeared in the protein profiles. The presence of common protein antigens was proven by Western blotting.     

Microchip gel electrophoretic analysis of perchloric acid‐soluble serum proteins in systemic inflammatory disorders

Perchloric acid (PCA) precipitation is a well‐known method for the separation of heavily glycosylated proteins and for reducing the masking effect of major serum proteins. The aim of this study is to characterize PCA‐soluble serum proteins in healthy individuals and in patients with systemic inflammatory diseases, such as Crohn's disease and sepsis. A PCA precipitation protocol was prepared and adapted to the analytical methods. After PCA treatment of the serum, the soluble proteins in the supernatant were analyzed by SDS‐PAGE and by microchip gel electrophoresis (MGE). Characteristic changes of the electrophoretic patterns of the PCA‐soluble fractions were observed. Four characteristic bands (at ～11, ～65, ～85, and ～120 kDa) with varying intensity were detected by MGE. The proportion of the ～65, ～85, and ～120 kDa bands were significantly higher in systemic inflammatory conditions than in healthy individuals (p < 0.001), and characteristic patterns were observed in patients with acute inflammation. The marked differences in the acid‐soluble protein patterns, which were observed in patients with ongoing systemic inflammation, might be a good indicator of inflammation. The MGE analysis is a fast screening and quantification method for the detection of characteristic changes among acid‐soluble serum proteins.     

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Integrated isotachophoretic stacking and gel electrophoresis on a plastic substrate and variations in detection dynamic range

In this study, we demonstrated an integrated ITP-gel electrophoresis (GE) device on a plastic substrate, in which 50 nL of samples could be hydrodynamically or electrokinetically injected and enriched by ITP into narrow bands and then subsequently introduced into a homogeneous GE channel for separation and detection. This microchip design rendered a simple introduction scheme for creating sandwiched stacking buffer system and flexibilities in choosing separation and stacking buffers independently. We used gel sieving buffers which compositions were different from those for stacking buffers to separate DNA and protein molecules based on sizing mechanism. Compared to conventional microchip GE, the sensitivity of microchip ITP-GE was estimated to increase by one to two orders of magnitude based on the dilution factor of the injected sample and the S/N ratio detected from the electropherogram. Moreover, it is interesting to note that ITP stacking leads to a preferential enhancement for analytes with lower concentrations compared to those with higher concentrations. Therefore, a reduction in the detection dynamic range for ITP-GE was gained. We demonstrated that ITP-GE could lead to 2-4-folds of reduction in the signal dynamic range for two PCR products in a mixture. Such advantage is demonstrated to be useful for the detection of two products amplified from a multiplex PCR in which one product is poorly amplified compared to the other.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

TMBETADISC-RBF: Discrimination of beta-barrel membrane proteins using RBF networks and PSSM profiles

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. We have developed a method based on radial basis function networks and position specific scoring matrix (PSSM) profiles generated by PSI-BLAST and non-redundant protein database. Our approach with PSSM profiles has correctly predicted the OMPs with a cross-validated accuracy of 96.4% in a set of 1251 proteins, which contain 206 OMPs, 667 globular proteins and 378 alpha-helical inner membrane proteins. Furthermore, we applied our method on a dataset containing 114 OMPs, 187 TMH proteins and 195 globular proteins obtained with less than 20% sequence identity and obtained the cross-validated accuracy of 95%. This accuracy of discriminating OMPs is higher than other methods in the literature and our method could be used as an effective tool for dissecting OMPs from genomic sequences. We have developed a prediction server, TMBETADISC-RBF, which is available at http://rbf.bioinfo.tw/~sachen/OMP.html.     

自由流电泳及其应用研究进展

对自由流电泳的分离原理、分离模式、影响分离的因素和条件以及自由流电泳仪器的发展进行了介绍,对近年来自由流电泳在离子、小分子和微粒分离,多肽和蛋白质分离,细胞和细胞器分离,药物对映体分离,微芯片装置以及蛋白质组学等方面应用研究的进展进行了综述。引用文献73篇。     

Protein separation using free‐flow electrophoresis microchip etched in a single step

A one‐step etching method was developed to fabricate glass free‐flow electrophoresis microchips with a rectangle separation microchamber (42 mm‐long, 23 mm‐wide and 28 μm‐deep), in which two glass bridges (0.5 mm‐wide) were made simultaneously to prevent bubbles formed by electrolysis near the Pt electrode from entering the separation chamber. By microchip free‐flow zone electrophoresis, with 200 V voltage applied, the baseline separation of three FITC labeled proteins, ribonuclease B, myoglobin and β‐lactoglobulin, was achieved, with resolution over 1.78. Furthermore, with 2.5 mM Na₂SO₄ added into the electrode buffer to form higher electrical field strength across separation microchamber than electrode compartments, similar resolution of samples was achieved with the applied voltage decreased to 75 V, which could obviously decrease Joule heat during continuous separation. All these results demonstrate that the free‐flow electrophoresis microchip fabricated by one‐step etching method is suitable for the continuous separation of proteins, which might become an effective pre‐fractionation method for proteome study.     

芯片毛细管电泳及其在生命科学中的应用

芯片毛细管电泳 (Chip CE)技术在近几年已取得了很大的进展。本文着重介绍芯片毛细管区带电泳技术 ,对等电聚焦、等速电泳、自由溶液电泳及胶束电动色谱等其它芯片电泳模式也有所提及。讨论了芯片材料和制作技术、芯片的几何形状、样品的操作和衍生、检测及芯片毛细管电泳技术的应用 ,特别是在核酸和蛋白质的分离分析中的进展     

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.     

Microchip electrophoresis-SDS methods with high-resolution and silver stain sensitivity for quality screening and quantitation of protein products

Two microchip electrophoresis (ME)-SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate-coated proteins within 1 min. In the high-resolution ME-SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen-binding (Fab) assemblies can be readily resolved and individually quantified. A high-sensitivity ME-SDS method was also developed with sensitivity comparable to that of SDS-PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low-expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high-resolution and the high-sensitivity ME-SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.     

一种可逆键合电泳微芯片的制作及在蛋白质分离中的应用

阐述了一种可逆键合电泳微芯片的制作方法, 以及电泳微芯片在蛋白质分离、临床尿蛋白检测方面的应用. 用标准光刻腐蚀技术在石英基片上腐蚀泳道, 清洗腐蚀好的基片和盖片后, 在真空条件下实现键合. 此种方法键合制作的电泳微芯片可重复键合使用, 制得的电泳微芯片成功地用于标准蛋白质分离以及临床尿蛋白分析.