毛细管等电聚焦分离蛋白质

采用均匀设计法进行未涂壁石英毛细管等电聚焦研究,考察两性电解质、甲基纤维素、四甲基乙二胺和牛磺酸几种组分对等电聚焦的影响,确定出了比较合适的浓度范围。当中性蛋白质迁移通过检测窗后,在进样端施加一低气压,从而使酸性蛋白质得到很好的分离,适用于较宽的ｐＩ范围。方法的重复性好,为蛋白质分离鉴定提供一种有力的工具。     

毛细管等电聚焦/加压毛细管电色谱二维分离体系在多肽分离中的应用

以毛细管等电聚焦(cIEF)为第一维分离模式,以反相加压毛细管电色谱(pCEC)为第二维分离模式,开展离线二维色谱分离研究,并对复杂肽段进行分离.羟丙基纤维紊(HPC)涂层的毛细管用于cIEF分离,对6种标准蛋白质的平均分离柱效约为31万.在毛细管末端引进电隔离槽,方便了第一维样品的收集.在加电6 kV下,第二维pCE...     

用于蛋白质分析的毛细管等电聚焦-电喷雾质谱接口的改进

蛋白质组学对其分析技术提出了大规模、高通量的要求 [1] .传统的等电聚焦 ( p I) -分子量 ( MW)双向电泳技术 ( 2 D- Gel)尽管在蛋白质组学研究中占有重要地位 ,但其操作繁杂、工作周期长 .Pandey等[1] 将毛细管等电聚焦 ( CIEF)与电喷雾质谱 ( ESIMS)联用 ,使得 p I和 MW两维分离鉴定技术变得简单迅速 .但 CIEF- MS的接口操作需中断高压和将毛细管阴极端插入电喷雾管 ,故引起分析蛋白质的散焦和不重现 .本工作改进了 CIEF- MS接口 ,采用毛细管阴极端和电喷雾针一体化的电喷雾接口 ,无需中断高压 ,实现了 CIEF- MS的在线联…     

毛细管等电聚焦/毛细管无胶筛分电泳二维蛋白质分离平台的构建

设计并制作了一种新型的中空纤维接口,以此为核心构建了毛细管等电聚焦(CLEF)／毛细管无胶筛分(CNGE)电泳二维蛋白质分离技术平台,实现了将二维凝胶电泳从平板转移到毛细管中。利用该二维分离平台,对血红蛋白样品进行了高效、快速分离分析,验证了该二维分离平台的可行性及分离效能。实验结果表明：二维分离系统总的峰个数和分离效能都比各自一维分离系统有较大提高。     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.

     

Factors Affecting the Performance of Capillary Isoelectric Focusing in Dynamically Coated Capillaries Using Polyethylene Oxide Polymer

Abstract

A simple, robust and reproducible capillary isoelectric focusing method using dynamically coated fused silica capillaries was developed. Simultaneous focusing and mobilization was successfully carried out using the hydrophilic polymer polyethylene oxide (PEO), which was added during sample preparation. The effects of preconditioning and anolyte and catholyte concentrations on the resolution and reproducibility of the method were optimized. Statistical screening of the significance of the effects of pressure, voltage, PEO concentration, and molecular weight on the efficiency of dynamic coating was carried out. Full-factorial design and analysis of variance (ANOVA) were used in order to determine the significance of each variable on the performance of the dynamic coat. Three response variables were selected to evaluate the method performance: migration time of a basic protein marker, migration time of an acidic protein marker, and difference in migration time between both markers. An empirically determined second order polynomial was employed to describe the relationship between the migration time and the isoelectric point under the experimental conditions studied in order to validate the method performance. The method was used under non-denaturing conditions to resolve a mixture of four standard proteins having isoelectric points covering a wide pH range (pH 3–11).     

Novel Methods for Controlling the Current During Isoelectric Focusing

While studying the effect of electrochemical reactions occurring at the electrodes on achievement of steady state in isoelectric focusing (IEF) we observed an abnormal increase of the current. Because the magnitude of the current determines the progress of IEF, knowledge gained from studies of its nature and generation may enable this effect to be controlled. We observed that addition of gelatin to the electrode solutions suppresses the magnitude of the current flowing through the system; this enables IEF to be performed under conditions closer to steady state, and steady state is achieved more quickly.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

毛细管柱表面鸡卵清蛋白快速改性并用于等电聚焦电泳分离蛋白质

开发了一种利用鸡卵清蛋白改性毛细管柱内表面的快速方法。改性后的毛细管柱可避免蛋白质样品的吸附作用，在较温和的条件下，柱性能有很好的稳定性，可适用于等电聚焦电泳分离生物样品的需要。     

Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.     

Analysis of Glycosylated Type II Interleukin-1 Receptor (IL-1R) by Imaged Capillary Isoelectric Focusing (i-cIEF)

Glycoproteins typically produce a complex charge profile due to their heterogeneous glycosylation pattern. We developed a reproducible imaged capillary isoelectric focusing (i-cIEF) method to monitor the charge variants of recombinant human Type II Interleukin-1 receptor (IL-1R), a heavily glycosylated protein expressed as a soluble receptor in Chinese Hamster Ovary (CHO) cells. This method was proved to be informative in multiple settings: monitoring of upstream process and downstream purification, analysis of in-process samples, characterization of the bulk drug substance, as well as testing of stability samples during drug development. The i-cIEF method was efficient at detecting changes in the charge isoform profile during different steps of the purification procedures or resulting from modifications of cell culture conditions. In addition, this method was well suited to monitor the consistency of sialic acid distribution and to detect deamidation events occurring in accelerated stability studies. The i-cIEF method presented here provides an improvement over IEF slab gels in terms of resolution, automation and quantitation. Due to its exquisite resolution of narrow pI range, this technology can find applications in quality control environment as identity assay as well as in the analytical laboratory to monitor subtle modifications of the protein.

     

Direct MALDI-MS Analysis of Proteins Isolated by Liquidphase Isoelectric Focusing Electrophoresis

A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then directly subjected to MALDI-TOF-MS analysis. By this approach, we are able to resolve two types of hemoglobins, A and C, which cannot be successfully separated by means of the traditional SDS-PAGE method.     

Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

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Carrier Ampholyte Isoelectric Focusing Based Two-Dimensional Electrophoresis in Preliminary Screening of Differential Proteomics Analysis

Two-dimensional electrophoresis is a current method for separating complex protein mixtures of a given sample in different states. In this study an improved carrier ampholyte isoelectric focusing method has been evaluated for its capacity for preliminary screening of expressional proteomics subjects. In comparison with current carrier ampholyte isoelectric focusing, this method showed enough resolution power to display major expressional changes in proteomic samples and demonstrated it can be used as a substitution for the immobiline based isoelectric focusing method.     

Electrochemical Reactions During Isoelectric Focusing and Their Role in Establishment of the pH Gradient

The process occurring as the background to IEF is electrolysis of water. During IEF the yield of water ions decreases, following a non-linear relationship similar to that of the current. Different extents of acidification and basification of the electrode solutions, causing a drift of the pH gradient, were observed. We derived a relationship underlying this process which enables calculation of the electrode current. By using a novel approach we showed that the sum of the pH of distilled water electrode solutions at the end of the IEF process tends to the ionic product of water.     

Synthetic Haptens and Monoclonal Antibodies to the Cyanotoxin Anatoxin‐a

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin‐a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin‐a, we have succeeded in generating the first‐ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin.     

毛细管等电聚焦-毛细管区带电泳二维蛋白质分离平台的构建及其性能

通常采用二维聚丙烯酰胺凝胶电泳 ( 2 D- PAGE)分析组织或细胞的全蛋白质 [1] ,但难与质谱 ( MS)直接联用 .用高效液相色谱 ( HPLC)和毛细管电泳 ( CE)分离分析蛋白质和多肽的一维分离模式的分辨率和峰容量有限 .多维柱联用技术比一维分离有更高的分辨率和峰容量 [2 ] ,便于和 MS直接联用 [3,4 ] ,易于实现自动化 .目前 ,有关 2 D- CE的报道相对较少 [5～ 7] ,我们初步实现了将 2 D- PAGE由平板转移到毛细管中[6 ,7] ,但凝胶柱的制作烦琐 ,存在交叉污染 ,不能与 MS直接联用 .本文用微透析中空纤维膜为接口构建了毛细管等电聚焦 ( …     

Oil/Alkanethiol Layers for the Study of Emulsified Protein Conformation by Surface Plasmon Resonance Using Monoclonal Antibodies

A method combining surface plasmon resonance and epitope mapping was developed to study the protein conformation at the oil/water interface of an emulsion. The conformation of beta-lactoglobulin stabilizing dodecane/water and miglyol/water interfaces was investigated using five anti-beta-lactoglobulin monoclonal antibodies. The developed method allows us to specifically recognize the emulsified beta-lactoglobulin at the surface of a sensor chip with good repeatability; i.e., standard deviations range between 0.7 and 3.6%. Considering that the monoclonal antibodies, recognizing conformational epitopes, still bind to beta-lactoglobulin at oil/water interfaces, it is concluded that the protein retains a globular conformation. It is shown that the inhibition-binding values of two pairs of Mabs are different for beta-lactoglobulin stabilizing dodecane/water and miglyol/water interfaces. This indicates that the conformations of emulsified beta-lactoglobulin are slightly different according to the nature of the oil phase. Copyright 2000 Academic Press.