Quantitative analysis of plasmid forms by agarose and capillary gel electrophoresis.

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.     

Enzyme-linked bridging assay method for the quantification of oligonucleotide-based drugs in biological matrices

With ongoing efforts to develop oligonucleotide-based (ODN-based) therapeutics, there is a need for a sensitive, high-throughput method of quantification of ODN-based drugs in biological matrices. To overcome the insufficient sensitivity and time-consuming sample extraction procedures involved in conventional capillary gel electrophoresis (CGE) and high-performance liquid chromatography (HPLC), we developed a nucleic acid hybridization-based enzyme-linked bridging assay (ELBA), which shows significant advantages over CGE methods in evaluating ODN-based drugs in plasma and tissue: (1) It has higher sensitivity; (2) it involves easier sample extraction procedures; (3) it is suitable for many ODN-based drugs, even those with different secondary structures and modifications, including phosphorothioate oligonucleotide (PSODN), mixed backbones with 2'-O-Me (MBO), locked nucleic acid (LNA) modifications, and B- and C-type CpG sequences; and (4) it is highly selective, even during simultaneous quantification, with regard to intact ODNs and their 3'-metabolites. This universal design produces a rapid, sensitive, specific assay with minimal method development time. It is well suited to high-throughput analysis of various ODN-based drugs.     

A nonradioisotope biomedical assay for intact oligonucleotide and its chain-shortened metabolites used for determination of exposure and elimination half-life of antisense drugs in tissue.

Rigorous extraction methods coupled with capillary gel electrophoresis (CGE) provide a basis for a nonradiolabel assay for quantitation of intact antisense drug and its numerous chain-shortened metabolites. As part of the validation of the CGE method, we compared the quantitation of unlabeled ISIS 3521 (ISI 641A) and its chain-shortened metabolites with total radioactivity of [(35)S]-ISIS 3521. ISIS 3521 was labeled on the fifth nucleotide linkage from the 5'-end with (35)S by well-established methods. Multiple tissues collected from rats after administration of [(35)S]-ISIS 3521 were assayed by both radiolabel (liquid scintillation spectroscopy) and CGE methods. The CGE method provided accurate quantitation of the drug and its metabolites in kidney cortex and liver tissues. The correlation between methods for multiple tissues over time was excellent with 88.5% of the measurements being statistically equivalent. These data suggest that CGE is an accurate means of quantitating oligonucleotide in tissue and that it compares favorably with traditional radiochemical techniques. Clearance half-lives for total measurable oligonucleotides were equivalent to clearance of total radioactivity in both liver and kidney with the longest clearance half-life associated with the kidney.     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.     

Identification and quantification of GC-rich oligodeoxynucleotides in tissue extracts by capillary gel electrophoresis

Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50cm neutrally coated capillary at 60 degrees C together with a 7M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions >or=1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.     

反义寡核苷酸药物癌泰得的定性分析方法研究

 癌泰得 (ACTCACTCAGGCCTCAGACT)为端粒酶表达抑制活性反义寡核苷酸 .为了探讨其定性检测手段 ,通过阴离子交换高效液相色谱和毛细管凝胶电泳分析方法 ,确定了该硫代寡核苷酸以及与其有关的短序列和部分未被硫代类似物的保留时间 ,并分析了不同混合物样品 .结果表明 ,阴离子交换高效液相色谱对硫代寡核苷酸骨架上的差异非常敏感 ,可很好地分离长度相同的硫代和未完全硫代类似物 ,并且随未被硫代磷酸基数目增加 ,保留时间依次缩短 .阴离子交换高效液相色谱对硫代寡核苷酸的长度不敏感 ,不能分离相差一个碱基的硫代寡核苷酸 .毛细管凝胶电泳可很好地分离长度相差一个碱基的硫代寡核苷酸 ,不能分离同长硫代和部分硫代寡核苷酸 .高效液相色谱结合毛细管凝胶电泳可有效地确定癌泰得的纯度和修饰程度 .     

Evaluation of microbial proteolysis in meat products by capillary electrophoresis

AIMS: The available methods for evaluating proteolysis in meat products, particularly the contribution of micro-organisms, are expensive, time-consuming and require an unacceptable sample size. To minimize these problems, two capillary electrophoresis-based methods have been developed. METHODS AND RESULTS: Six Gram-positive, catalase-positive cocci, four moulds and three yeasts, isolated from dry-cured ham, were tested on sterile pork slices. Using the Capillary Gel Electrophoresis (CGE) method, changes in sarcoplasmic and myofibrillar proteins due to endogenous and microbial enzymes were detected. The Capillary Zone Electrophoresis (CZE) analysis allowed evaluation of bulk changes by micro-organisms in soluble nitrogen compounds. CONCLUSION: CGE analysis of myofibrillar proteins and CZE determination of soluble nitrogen compounds have proved to be valuable tools for evaluating proteolytic activity of endogenous and microbial origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The CGE and CZE methods developed can be used for a rapid and sensitive analysis of proteolysis in meat products.     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

A new approach for on-line enrichment in electrophoresis of dilute protein solutions

A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.     

不同富集和分离培养条件下的含酚废水处理生物膜微生物群落结构与活性比较分析

采用Biolog和变性梯度凝胶电泳(DGGE)技术研究了不同苯酚浓度培养对焦化废水处理厂反硝化池生物膜样品中微生物群落结构和代谢类型的影响。DGGE结果表明, 不同浓度苯酚和不同培养方式富集培养后, 细菌16S rDNA的部分条带分布谱形发生改变, 还有部分条带只受到了苯酚浓度变化的影响; 富集培养过程中由于碳源组成相对焦化废水简单, DGGE条带所代表的优势微生物多样性有所降低。Biolog试验结果表明, 生物膜样本的细菌群落代谢能力最强; 低浓度苯酚富集后的样品能利用的底物碳源类型最丰富。对Biolog试验结果的主成分分析显示, 相同浓度苯酚富集培养后的细菌群落代谢功能多样性相似, 但从DGGE结果看出其结构组成产生了变化。富集培养使样品微生物群落的代谢功能发生改变, 低浓度的苯酚富集增加了群落中微生物的代谢类型。而不同条件获得的分离物其苯酚降解能力的初步分析也表明, 富集与分离条件对苯酚降解菌的分离能力和得到的菌株特性具有差别。     

Assessment of sample preparation methods for metaproteomics of extracellular proteins

Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.     

Alternative and effective proteomic analysis in Arabidopsis

Various functional genomics platforms are required to define the phenotype associated with a mutant. Global protein analyses may be included in any study. We describe here a rapid method of protein sample preparation and analysis, suitable for all laboratories and using Arabidopsis plantlets as the starting material. This reliable and reproducible method for high yield protein extraction from small amounts of material can be used on even the most recalcitrant tissues. The proteins extracted are suitable for many types of protein analysis, including nondenaturing investigations. This method was validated by a rigorous 2-DE approach, coupled with unambiguous LC-MS/MS identifications featuring strong sequence coverage (average of 26% with eight different peptides/spot protein). The reproducibility of the method was demonstrated by multiple protein identifications from identical series of spots. An interactive map (http://www.isv.cnrsgif.fr/gel2d/), including 435 protein variants showed that (i) 38% of the proteins were yet unreported, (ii) reduced subfractionation, (iii) had frequent protein modifications (average of two spots/protein entry), and (iv) underwent no major proteolytic events other than leader peptide cleavage. Finally, a simple mobility shift method for the large subunit of RuBisCo (LS) in the first dimension made it possible to characterize previously masked protein spots.     

Alternative methods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations

Current methods for the study of pigments involve freezing in liquid nitrogen and storage at −80°C or lyophilization until HPLC analysis. These requirements greatly restrict ecophysiological research in remote areas where such resources are hardly available. We aimed to overcome such limitations by developing several techniques not requiring freezing or lyophilization. Two species with contrasting foliar characteristics (Olea europaea and Taraxacum officinale) were chosen. Seven preservation methods were designed, optimized and tested in a field trial. These protocols were compared with a control immediately frozen after collection. Pigments and tocopherols were analysed by HPLC. Main artefacts were chlorophyll epimerization or phaeophytinization, carotenoid isomerization, altered de-epoxidation index and tocopherol degradation. Among all methods, sample desiccation in silica gel provides robust samples (pigment composition was unaffected by storage time or temperature) and almost unaltered pigment profiles, except for a shift in epoxidation state. Although liquid nitrogen freezing and subsequent lyophilization or freezer storage were preferred, when these facilities are either not available or not suitable for long-distance transport, desiccation with silica gel, passive extraction in acetone and/or storage of fresh samples in water vapour saturated atmospheres enable a complete pigment characterization. Silica gel is advisable for long-term sample conservation.     

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.     

Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.     

鲍曼不动杆菌烈性噬菌体的分离与纯化

利用柱层析方法,纯化鲍曼不动杆菌（Acinetobacter baumannii）烈性噬菌体AB1。首先采用聚乙二醇6000沉淀方法,初步分离裂解液中的噬菌体,噬菌体纯度由6.1×1010 pfu/mg提高到37×1010 pfu/mg,噬菌体回收率为58.8%,蛋白质去除率为90.6%;噬菌体粗提样品经Sepharose 4B凝胶过滤层析柱进一步纯化,纯度提高到73×1010 pfu/mg,噬菌体回收率为95.7%,蛋白质去除率为48.1%;收集的噬菌体样品最后经DEAE-52阴离子交换层析柱处理,噬菌体纯度为40×1010 pfu/mg,回收率为50.8%,蛋白去除率15.6%。内毒素分析结果显示,Sepharose 4B凝胶过滤层析纯化的噬菌体样品中,内毒素含量为443.8 EU/mg,而DEAE-52阴离子交换层析纯化的噬菌体样品中,内毒素含量为544.4 EU/mg。实验结果显示,PEG沉淀方法与Sepharose 4B凝胶过滤方法能够有效地提高噬菌体纯度,而DEAE-52阴离子交换层析则不能提高噬菌体的纯度,也无法有效地去除样品中的内毒素。     

Development and optimization of two-dimensional-electrophoresis protocol ofLeptospirillum ferriphilum

Leptospirillum ferriphilum is important in bioleaching, in which process it is often under heavy stresses of heavy metal ions and high oxidation reduction potential (ORP). Two-dimensional-electrophoresis (2-DE) and comparative proteomic analysis are useful to investigate the responses ofL. ferriphilum to environmental stresses. But, 2-DE analysis forL. ferriphilum is not successful as the samples ofL. ferriphilum contain low protein concentration, complex composition, high salt concentration, and many other interfering components, which make it difficult for 2-DE analysis. In this research, optimizations on the sample preparation and purification methods, sample volume, sample loading methods for isoelectric focusing (IEF), and gel visualization methods were made. More than 629 Coomassie stained spots in single gel were obtained. The image quality and protein concentration in most of the spots met the requirements for both differential spots analysis and mass-spectrum analysis. The 2-DE protocol forL. ferriphilum was successfully developed for the first time.     

Comparison of Emulsifying Properties of Plant and Animal Proteins in Oil-in-Water Emulsions: Whey,Soy, and RuBisCo Proteins

There is increasing interest within the food industry in replacing animal-derived ingredients with plant-derived alternatives. In this study, we compared the emulsifying properties of an emerging plant protein (RuBisCo protein) with those of a well-established plant (soy protein) and animal (whey protein) protein. The RuBisCo protein (ribulose 1,5-bisphosphate carboxylase) was isolated from duckweed (lemna minor), which is an abundant plant material with a higher protein yield and biomass per unit area than most other plant protein sources. The ability of the three proteins to form and stabilize 10 wt% soybean oil-in-water emulsions was examined. The minimum amount of protein required to produce small droplets (d <?350 nm) decreased in the following order: RuBisCo > soy > whey protein. This effect was mainly attributed to the fact that the molar mass of the proteins decreased in the same order. Even so, the RuBisCo proteins were able to form stable emulsions when used at sufficiently high concentrations (≥ 1%). All three types of protein-coated oil droplets aggregated at pH values near their isoelectric points and at high ionic strengths but there were differences between them. In the absence of added salt, extensive droplet aggregation occurred from pH 4 to 5 for whey protein, from pH 2 to 5 for soy protein, and from pH 2 to 6 for RuBisCo protein. The isoelectric points of all three protein-coated droplets were around pH 5, but the magnitude of the surface potential at low and high pH values was higher for whey protein than for the two plant proteins. At pH 7, extensive droplet aggregation occurred at ≥100 mM NaCl for RuBisCo- and soy protein-coated droplets, but only at ≥400 mM NaCl for whey protein-coated ones. The RuBisCo-coated oil droplets were more prone to flocculation when heated, especially in the presence of salt (100 mM NaCl). Overall, these results show that RuBisCo protein can be used to form and stabilize oil-in-water emulsions, but the pH, salt, and temperature conditions must be carefully controlled to avoid droplet aggregation. We should note that droplet aggregation is advantageous in some applications because it leads to an increase in emulsion viscosity or gelation.

     

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.     

Fast molecular diagnostics of canine T-cell lymphoma by PCR and capillary gel electrophoresis with laser-induced fluorescence detector

Lymphoma is the most common hematopoietic tumor in dogs and manifests as a proliferation of malignant lymphoid cells primarily affecting the lymph nodes or solid visceral organs. We describe the use of capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector based on polymerase chain reaction (PCR) to rapidly detect a disorder of the canine T-cell receptor gamma (TCRgamma) gene. After the PCR amplification of the specific TCR( gene in dogs, the 90-bp DNA fragment amplified was separated in a fused-silica capillary by CGE-LIF. Under an electric field of 375 V/cm and with a sieving matrix of 1.5% poly (ethyleneoxide) (M(r) 600,000), the amplified PCR products were analyzed within 4 min by CGE separation. When the CGE-LIF method was applied to real clinical samples of the specific DNA fragment of the TCR( gene, the migration time and the corrected peak area showed relative standard deviations (n=5) of 0.29% and 0.58%, respectively. Both methods of CGE-LIF and slab gel electrophoresis showed same results for nine clinical samples. This PCR/CGE-LIF technique may prove to be a new fast and simple tool for the rapid diagnosis of the PCR-amplified DNA of canine T-cell lymphoma.