Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking

The paper describes a method of size separation of proteins by capillary sieving electrophoresis with cationic surfactant. Proteins are separated within 12 min with repeatability of migration times better than 0.2%. Some proteins achieve the separation efficiency of 200,000 theoretical plates. The method can be used for determination of protein relative molecular masses. The accuracy of the determined relative molecular masses and the limitation of the method were investigated by the analysis of more than 60 proteins. The method also allows separation of protein oligomers. Proteins can be quantitated after the electrokinetic injection in the concentration range 0.07-0.43?g/L. The average detection limit is about 2?mg/L.     

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.     

Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell.

Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated by using 8% pullulan as the sieving matrix. This method offers a few advantages for single-cell protein analysis. First, it provides reproducible separation of single-cell proteins according to their size. Based on comparison with the migration time of standard proteins, most components from a single HT29 cancer cell have molecular masses within the range of 10-100 kDa. Second, as a one-dimensional separation method, it gives fairly good resolution for proteins. Typically, around 30 protein components of a single HT29 cell were resolved, indicating that this method has similar peak capacity to SDS-polyacrylamide gel electrophoresis (PAGE). Third, this method shows high detection sensitivity and wide dynamic range, which is important because of the wide range of protein expression in living systems. Detection limits for standard proteins ranged from 10(-10) to 10(-11) M. Finally, this method provides much higher speed than classical gel electrophoresis methods, and it provides automated anlysis of cellular proteins at the single-cell level; the separation is complete in 30 min and the entire analysis takes approximately 45 min.     

几种标准蛋白质的毛细管区带电泳的分离及其迁移行为

本文采用毛细管区带电泳分离了几种标准蛋白,研究了不同的清洗方式,不同ＰＨ的缓冲溶液等对迁移行为及分离的影响,并同时考察了迁移时间和相对迁移时间与操作电压的关系。     

Characterization of glycerin-based polyols by capillary electrophoresis

Methods based on capillary electrophoresis (CE) have been developed to obtain the molar mass distribution (MMD) of glycerin-based polyols and details on the presence of mono- and difunctional byproducts in technical samples. Prior to the analyses the hydroxy end-groups of the trifunctional polyols were converted to chargeable and UV-active moieties with phthalic anhydride (PhAH) as the derivatization reagent. With a method of capillary zone electrophoresis (CZE) samples of glycerin-based polyols with average molar masses up to 6000 were separated according to their charge-to-size ratio. The separations were carried out with a buffer solution containing 50% (v/v) acetonitrile and 10 mM sodium tetraborate, and for detection UV absorption at 220 nm was measured. An approximately linear relation between the reciprocal of the effective mobilities and the degree of polymerisation of the glycerin-based polyols was found. Therefore, the proposed CZE system could be used to determine the degree of polymerisation and polydispersity of technical glycerin-based polyol samples. The effect of the presence of sodium dodecyl sulfate (SDS) in the buffer solution on the CE separation of linear polyethylene glycols (PEGs), polypropylene glycols (PPGs) and ethylene oxide-propylene oxide (EO-PO) copolymers with different molar masses was investigated. The interaction between the charged polymer derivatives and SDS ions in solution increased strongly with the degree of polymerisation and the amount PO in the chain of the polymeric compounds. This behaviour made it possible to invert the migration order of EO-PO containing polymers of different size. With a background electrolyte (BGE) composition of 10mM SDS and 25% (v/v) acetonitrile in borate buffer mono- and difunctional byproducts were separated from the main glycerin-based polyols based on their number of end-groups. Accurate quantities for the mono- and difunctional impurities in technical glycerin-based polyol products were determined.     

Ultra-fast high-performance capillary sodium dodecyl sulfate gel electrophoresis of proteins

An ultra-fast analysis of proteins, based on sodium dodecyl sulfate (SDS)-mediated gel electrophoresis was developed, in which protein molecular mass standards ranging from M_r 14 200 to 94 700 were separated within 3 min. A 50 μm diameter uncoated fused-silica capillary column and a high field strength are used. The effects of the SDS concentration in the separation gel buffer and in the sample buffer on the resolution of protein test mixture were studied. The influence of the heat treatment of the sample prior analysis is also discussed.     

Capillary electrophoresis of seed 2S albumins from Lupinus species

Two modes of capillary electrophoresis (CE)--free-solution capillary zone electrophoresis (CZE) and sodium dodecyl sulfate capillary electrophoresis (SDS-CE) using a non-gel sieving matrix--have been developed for comparative analysis of low-molecular-mass 2S albumin isoforms from lupins. The albumin fraction and 2S albumins were separated in uncoated fused-silica capillary by CZE with 0.02 M phosphate buffer, pH 7.3, containing the sodium salt of phytic acid. The use of phytic acid (0.025 M) as buffer modifier and ion-pairing agent improved migration reproducibility, peak shape and separation efficiency. The reduced 2S albumins were separated by SDS-CE using a high concentration (0.3-0.5 M) mixture of tris(hydroxymethyl)aminomethane and borate buffers in uncoated fused-silica capillary. Of the various polymers used as non-gel sieving matrix, SDS-CE with a 10% dextran solution was found to be suitable for separation of 2S albumin polypeptides with molecular masses of 4,000-7,000 and 8,000-11,000. The addition of glycerol or ethylene glycol to the SDS separating buffer improved the resolution of polypeptides. The examined Lupinus species showed species-specific CZE and SDS-CE migration profiles of the 2S albumins.     

Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.     

玻璃微流控芯片十二烷基硫酸钠无胶筛分电泳测定免疫球蛋白片段分子量

通过实验优化了葡聚糖筛分介质和运行缓冲溶液的浓度,采用十二烷基硫酸钠(SDS)无胶筛分电泳分离体系(10%(w/v)葡聚糖,0.1%SDS,10%甘油,0.2mol/LTris-硼砂,pH8.3的缓冲液)在自制的玻璃微流控芯片上高效分离了BODIPY衍生的蛋白质分子量标准样品,连续6次电泳所得迁移时间的相对标准偏差均小于0.50%。以6种蛋白质分子量的对数对其迁移时间作图,线性回归良好(r=0.994)。采用该芯片电泳分析体系对免疫球蛋白G不同片段的分子量进行了测定,所得结果与实际基本相符。     

CSE-MECC two-dimensional capillary electrophoresis analysis of proteins in the mouse tumor cell (AtT-20) homogenate

Two-dimensional capillary electrophoresis was used for the separation of proteins and biogenic amines from the mouse AtT-20 cell line. The first-dimension capillary contained a TRIS–CHES–SDS–dextran buffer to perform capillary sieving electrophoresis, which is based on molecular weight of proteins. The second-dimension capillary contained a TRIS–CHES–SDS buffer for micellar electrokinetic capillary chromatography. After a 61-s preliminary separation, fractions from the first-dimension capillary were successively transferred to the second-dimension capillary, where they further separated by MECC. The two-dimensional separation required 60 min.     

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.     

Microemulsion electrokinetic chromatography of proteins.

Microemulsion electrokinetic chromatography was used to separate a test mixture of proteins effectively. The separation was carried out in a 42.5 cm (to the detector) x 50 microns I.D. fused-silica capillary using a microemulsion system consisting of 80 mM heptane, 120 mM SDS, 900 mM butanol in 2.5 mM borate buffer, pH 8.5-9.5. Optimum separation conditions were investigated with respect to the running voltage, temperature, pH and the composition of microemulsion. Results were compared with those obtained in micellar electrokinetic chromatography and capillary zone electrophoresis. The examined method is practical and successfully applied to the assay of genetically engineering pharmaceuticals, recombinant human granulocyte macrophage colony stimulating factor injection and recombinant human granulocyte colony stimulating factor injection.     

Capillary electrophoresis of proteins for proteomic studies

Analyses of proteins in complex mixtures such as cell lyzates are presently performed mainly by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. For structural analysis, each protein in a spot is digested with proteases and the fragment peptides are subjected to Edman sequencing and/or mass spectrometry. These works aim at the total analysis of proteins in a complex mixture and reconstruction of their cooperative functions. Genomic studies are now being combined with these proteomic studies. This review article focuses on the application of capillary electrophoresis aiming at the total analysis of complex protein systems or structural analysis of each separated protein. From this viewpoint, articles on capillary zone electrophoresis, capillary isoelectric focusing, and sieving SDS capillary electrophoresis are reviewed. Since these techniques of capillary electrophoresis have been thoroughly reviewed previously, papers published in 1997 and 1998 are mainly covered.     

Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.     

胶束电动毛细管色谱法测定消糖灵中的优降糖

 以甲氧苄氨嘧啶为内标,采用胶束电动毛细管色谱法分离测定了新药消糖灵中的优降糖。电泳条件：以25 mmol/L硼砂-30 mmol/L十二烷基硫酸钠（pH 9.0）为电泳介质,未涂层石英毛细管（50 μm i.d.×39.5 cm,有效分离长度34.8 cm）为分离通道,压力进样(68.95 kPa.s),17 kV恒压电泳（28 ℃）,检测波长228 nm。优降糖在14 min内与其他成分得到很好分离,且质量浓度为25 mg/L～275 mg/L时,优降糖可进行定量分析,加标回收率为(100.6±1.4)%。方法简便、快速,结果准确,重现性好,可用于优降糖复方制剂的质量控制。     

Chemical modification of proteins to improve the accuracy of their relative molecular mass determination by electrophoresis

We studied the electrophoretic behavior of basic proteins (cytochrome c and histone III) and developed a carbamylation method that normalizes their electrophoretic size separation and improves the accuracy of their relative molecular mass determined electrophoretically. In capillary zone electrophoresis with cationic hitchhiking, native cytochrome c does not sufficiently bind cationic surfactants due to electrostatic repulsion between the basic protein and cationic surfactant. Carbamylation suppresses the strong positive charge of the basic proteins and results in more accurate relative molecular masses.     

毛细管电泳-激光诱导荧光检测法分析胃癌组织及癌旁正常组织蛋白质的差异性

胃癌是临床常见的恶性肿瘤之一。近年来寻找肿瘤相关特异蛋白质是蛋白质组学研究的热点。本文通过考察毛细管动态涂层方法、筛分介质聚环氧乙烷(PEO)的浓度、缓冲液的pH值、分离电压、温度及荧光染料对分离效果的影响,建立了毛细管电泳-激光诱导荧光法分离胃癌组织及癌旁正常组织蛋白质的方法;通过分离检测,获得两者的蛋白质指纹图谱。经分析,两者的指纹图谱相似度达到0.8以上,差异蛋白质分子质量集中在50000~100000 Da之间,提示某些小分子蛋白质可能是和肿瘤发生相关的特异蛋白质,从而缩小了特异性分子标记物的筛选范围。病理组织学分型及蛋白质电泳峰数目的统计结果验证了该方法的可靠性。该方法具有临床应用的潜力。     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

High-performance capillary electrophoresis of proteins. Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins.

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship (r greater than 0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10,000-100,000 and 40,000-200,000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 X 10(5)-1 X 10(6) per metre are routinely being obtained. Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 0.999) and the proteins may be quantified with an R.S.D. of ca. 3-7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound (ca. 60,000 dalton) may be differentiated by HPCE with a gel-filled capillary column.     

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 microm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of -12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.