亲和毛细管电泳技术及其应用

对近几年新发展起来的亲和毛细管电泳技术（ACE）的原理、分类及方法作了简要介绍,着重介绍了亲和毛细管区带电泳、毛细管亲和凝胶电泳、胶束电动色谱中的亲和电泳、亲和毛细管等电聚焦、亲和探针毛细管电泳等过程和方法。对ACE在分子生物学、生物化学中的应用及该技术在亲和常数测定、核酸片段识别、竞争免疫分析、药物先导化合物的筛选等方面的应用也作了介绍。     

亲和毛细管电泳技术在蛋白质-DNA相互作用研究中的应用

蛋白质-DNA的相互作用在决定细胞命运的许多过程中发挥重要作用,对蛋白质-DNA相互作用的分子机制研究有利于对基本生命过程的理解,为相关疾病的临床治疗及药物筛选提供理论指导。另一方面,利用一些已知的蛋白质-DNA相互作用可以帮助开发先进的生物工程和生命分析技术,为相关研究提供有力的技术支持。因此,建立灵敏、快速的分析方法用于表征蛋白质-DNA的相互作用十分重要。高效毛细管电泳（capillary electrophoresis,CE）技术因其超高的分离效率、极低的样品消耗与较短的分析时间等优势被广泛应用于化学、生命科学和环境科学等多个研究领域。其中,亲和毛细管电泳（affinity capillary electrophoresis,ACE）技术已经成为考察分子间相互作用的重要研究工具。这篇文章综述了亲和毛细管电泳技术自建立以来在蛋白质-DNA相互作用分析方面的研究进展,并对经典的研究工作进行了着重介绍,主要包括三方面的内容：（1）亲和毛细管电泳技术简介;（2）利用亲和毛细管电泳技术进行蛋白质-DNA相互作用的基础分子机制研究;（3）利用已知的蛋白质-DNA相互作用发展针对目标分子及目标反应的亲和毛细管电泳检测技术。本文还对该领域的未来发展趋势进行了展望与探讨,提出应从以下两个方面增强亲和毛细管电泳技术的分析能力：（1）充分发挥CE技术样品消耗少和高通量等优势,分别发展针对少量珍贵生物样品的高灵敏检测方法和针对大量未知因素的高通量筛选方法;（2）结合DNA测序及质谱技术快速筛选、鉴定未知的蛋白质-DNA相互作用的精确靶点。     

毛细管电泳在生物大分子分析中的应用研究进展

近年来,毛细管电泳技术以其高效、微量、分离模式多样等优势,在生物大分子分析领域得到了日益广泛的应用.综述了毛细管电泳技术在多糖、蛋白质、核酸和脂质中的应用,并对其相关的样品制备、毛细管内壁吸附、仪器的研制等方面存在的问题进行分析和展望,以期为毛细管电泳技术在生物大分子领域的推广应用提供借鉴.     

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     

芯片国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。     

毛细管电泳技术在重大疾病特异性蛋白质分析中的应用

人类重大疾病特异性蛋白质的识别、鉴定等是目前生命科学亟待解决的问题。毛细管电泳技术用于蛋白质分析,具有装置简单、分析速度快、试剂和样品消耗少以及有多种分离模式和检测手段等特点。本文综述了毛细管电泳技术在人类重大疾病特异性蛋白质分析中的应用,包括肿瘤疾病、神经退行性疾病和输血性传染病等的特异性蛋白质检测。     

毛细管电泳质谱联用技术及其应用

本文介绍了用于毛细管电泳质谱联用仪器的多种接口技术，描述了ＣＺＥ，ＣＩＥＦ，ＣＧＥ，ＭＥＫＣ和ＣＩＴＰ等毛细管电泳技术和四极质谱，离子阱质谱，傅 叶变换离子回旋共振质谱，飞行时间质谱，磁质谱，解吸质谱等联用的现状及发展前景，对近年来ＣＥ－ＭＳ在酶解产物。蛋白质和肽，核苷酸，药物及代谢产物等领域中的应用作了详细述评。     

高效毛细管电泳在核酸、蛋白质分析中的新进展

高效毛细管电泳以其分离效率高，分析速度快，样品和试剂用量少，易于实现自动化等优点，在核酸、蛋白质等生物样品的分析方面发挥着重要的作用并具有巨大的潜力。本文介绍了近两年来高效毛细管电泳技术的进展，特别是PCR／CE、CE／MS以及电泳芯片技术等方面的新发展，并综述了高效毛细管电泳在核酸、蛋白质分析方面的应用，同时对其前景进行了展望。     

毛细管电泳技术在氨基酸分析中的研究进展

对毛细管电泳技术在氨基酸分析中的研究进展进行了综述,分析了直接法和衍生法对氨基酸进行分析的优缺点,详细叙述了毛细管电泳中的紫外、激光诱导荧光、电化学及质谱等检测方法在氨基酸分析中的应用,并重点总结了毛细管电泳在手性氨基酸分离中的应用.     

核酸适配体亲和色谱的研究进展

核酸适配体亲和色谱是将核酸适配体作为色谱固定相上的亲和配体的一种新型色谱技术。核酸适配体是一种可以特异性地识别目标物的寡聚核苷酸,与免疫抗体相比,核酸适配体在筛选制备、稳定性及应用等方面都显示出独特的优点。本文介绍了核酸适配体亲和色谱在小分子、蛋白质和细胞的分离和分析中的应用,对核酸适配体亲和色谱的研究现状和发展前景进行了综述。     

Identification, quantitation, and characterization of biomolecules by capillary electrophoretic analysis of binding interactions

The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis.     

色谱技术在药物-血浆蛋白相互作用研究中的应用进展

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α₁ -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。     

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×10⁹) and binding constant (K_b = 1×10⁴ L/mol) of the interaction between RAW264.7 and naringin.     

Affinity CE Determination of the Binding Constant of Bioactive Sulfated Polysaccharide 916 to Human Serum Albumin

Drug-protein binding is an important process in determining the activity of a pharmaceutical agent once it has entered the body. This paper developed an affinity capillary electrophoresis method to determine the binding constant between a bioactive sulfated polysaccharide 916 (916) and a potential protein, human serum albumin. This method is based on the principle that the changing analytes have different mobility shift in the zone electrophoresis. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant of 916 to the HSA complex. The binding constant Ka of 916 to the human serum albumin achieved was 2.1 × 10⁴.     

Determination of the binding parameters for antithrombin-heparin fragment systems by affinity and frontal analysis continuous capillary electrophoresis

The suitability of affinity capillary electrophoresis (ACE) and frontal analysis continuous capillary electrophoresis (FACCE) for binding constant determination was investigated for complexes between heparin fragments and antithrombin III, one of the main target proteins in the coagulation cascade. In a 100 mM ionic strength phosphate buffer (pH 7.4), ACE was suitable to determine weak to medium interactions developed by short oligomeric heparin fragments, but it failed for decasaccharide, which presents a more complex irreversible interaction. However FACCE allowed evaluating the binding constant for these longer oligomeric fragments. Both experimental approaches were complementary for a wide variety of heparinic fragments.     

On-line post-capillary affinity detection of immunoglobulin G subclasses and monoclonal antibody variants for capillary electrophoresis

Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-₁-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.     

Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis

A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.     

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.     

Capillary electrophoretic methods applied to the investigation of peptide complexes

This article gives an overview of the applications of capillary electrophoretic methods to investigate the non‐covalent interactions of peptides (peptide complexes) with variable middle‐ and high‐molecular‐mass receptors (ligands) as well as with small ions and molecules in the period 2007–2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel–Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.