载液组成对于卵白蛋白在非对称流场流分离通道中膜吸附和聚集行为的影响

非对称流场流分离技术对于蛋白质等生物大分子的分析具有温和、分离范围广的特点。然而,在场流分离通道中,受载液组成的影响而产生的蛋白质与通道膜的相互作用和蛋白质在通道内的聚集行为,会影响分析物的回收率和尺寸形态,这些现象一定程度上限制了场流分离仪器的进一步应用。该文研究了载液组成对于卵白蛋白在非对称流场流分离中膜吸附和聚集行为的影响。考察了不同pH （6.2、7.4、8.2）、阳离子种类（Na⁺、K⁺、Mg²⁺）及多种离子强度（0~0.1 mol/L）等条件对卵白蛋白洗脱过程的影响。结果表明a）载液的离子强度越大,卵白蛋白的吸附和聚集行为越严重;b） pH和蛋白质的等电点pI的相对大小决定了蛋白质的表面电荷,从而影响蛋白质的吸附聚集行为;c）二价阳离子Mg²⁺更易引发通道中蛋白质的吸附和聚集。这些结果有助于今后使用非对称流场流分离技术分析蛋白质样品时,改善载液组成以获得更高的回收率,降低蛋白质聚集作用,对AF4更广泛地应用于蛋白质生化分析中有较好的参考价值。     

免疫亲和质谱法研究／β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法,对β2-微球蛋白抗原表位进行了系统研究.完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后,用Endoproteinase Glu-C,Trypsin,AminopeptidaseM和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分了,并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究.结果表明:β2-微球蛋白抗原表位位于整个蛋白分了氨基酸序列的61～67位,即为SFYLLYY.通过合成肽段的分析,证明了SFYLLYY即为抗原表位,与亲和质谱方法分析结果一致.     

4-巯基苯硼酸修饰二维二硫化钼纳米复合材料的制备及其用于N-糖肽特异性富集

蛋白质的N-糖基化修饰在多种生物过程中发挥着至关重要的作用，近年来的许多研究证实异常的蛋白质糖基化与多种疾病的发生发展密切相关，表明糖基化蛋白质具有较大的潜力成为新的生物标志物或者药物靶标。在样本的处理过程中，对N-糖基化肽段进行富集分离后再进行质谱分析已经成为糖蛋白质组学分析前的必要步骤。但是，由于复杂生物样本中N-糖基化肽段的丰度低和离子化效率差等问题，通过质谱鉴定N-糖肽仍然是一项艰巨的任务。研究通过将纳米金线（Au）、4-巯基苯硼酸（4-MPB）与超薄二维二硫化钼（2D-MoS₂）进行反应，成功制备了一种用于富集蛋白质N-糖基化肽段的新型功能纳米复合材料（MoS₂/Au/4-MPB）。二硫化钼纳米材料的层状结构可以为反应提供大量的可修饰位点，便于修饰纳米金线；功能基团4-巯基苯基硼酸对N-糖肽具有高度的选择性，可以对生物样品中N-糖基化肽段进行特异性富集。使用标准蛋白人免疫球蛋白G（IgG）和牛血清白蛋白（BSA）胰蛋白酶酶切产物对新型功能纳米材料的N-糖基化肽段的富集性能进行评估，其灵敏度达到5 fmol，选择性达到1：1000。将其用于生物样品中N-糖基化肽段的富集，从50 μg尿液外泌体蛋白胰蛋白酶酶切产物中共富集鉴定出768个N-糖肽，归属于377个蛋白质。这些结果表明该新型功能纳米复合材料对复杂生物样品中N-糖肽的选择富集有着巨大的应用潜力，为糖蛋白质组的研究提供了一种新方法。     

高效液相色谱-串联质谱法测定肉制品和调味料中7种水产品过敏原

基于高效液相色谱-串联质谱系统建立了食品中水产品过敏原的快速筛查和定量检测方法。样品经蛋白质提取、纯化、胰蛋白酶解后，利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）结合ProteinPilot软件，基于母离子和碎片离子谱分析，实现蛋白质和多肽的鉴定。再通过基本局部比对搜索工具（BLAST）与Uniprot数据库对比分析，筛选出南美白虾、大闸蟹、青蟹、金枪鱼、大西洋鲑鱼的7种过敏原蛋白的30个特征肽。利用高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）系统对特征肽进行验证和多反应监测（MRM）定量研究。结果表明，该方法在5~250 mg/kg范围内线性关系良好，检出限为2~3.5 mg/kg，平均回收率为88.7%~110.2%。该方法重现性好，通量高，可应用于肉制品和调味料中7种过敏原的快速筛查和定量分析。     

免疫亲和质谱法研究β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法, 对β2-微球蛋白抗原表位进行了系统研究. 完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后, 用Endoproteinase Glu-C, Trypsin, Aminopeptidase M和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分子, 并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究. 结果表明: β2-微球蛋白抗原表位位于整个蛋白分子氨基酸序列的61～67位, 即为SFYLLYY. 通过合成肽段的分析, 证明了SFYLLYY即为抗原表位, 与亲和质谱方法分析结果一致.     

大豆主要过敏原Gly m Bd 30K抗原决定簇表征预测研究

本文应用生物信息学技术,通过使用三种软件SOPMA、Swiss-model和DNAStar来预测大豆主要过敏原Gly m Bd 30K的抗原表位,结果发现80-85、103-106、217-220、355-360这四段氨基酸残基序列是可能的抗原表位,为后续的蛋白表位实验提供了依据,大大提高了工作效率.     

肽段固相等电聚焦结合液相色谱-质谱联用技术分析鼠肝蛋白质

利用肽段固相胶条等电聚焦技术对大鼠鼠肝组织蛋白质酶解产物进行预分离,反相色谱分离所得预分离组分后,用LTQ-Orbitrap MS进行分析。共鉴定到2039个蛋白质,包括18个乙酰化蛋白,其中4个乙酰化蛋白未被报道过。对这些蛋白进行了生物信息学分析。结果表明,将肽段等电聚焦与LC-MS/MS结合起来,是一种有效的蛋白质组学分析技术,适合于大规模蛋白质鉴定分析。     

疑似蛇毒液样品的纳升级超高效液相色谱-高分辨质谱分析鉴定

针对5个疑似蛇毒毒液及其沾染样品,基于纳升级超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（Nano LC-MS/HRMS）技术,结合尺寸排阻色谱分离,建立了一种蛋白质种类及物种归属的严格鉴定方法。5个样品经尺寸排阻色谱分离后均得到3个洗脱峰,分别冻干后以胰蛋白酶进行溶液内酶解处理并进行液相色谱-高分辨质谱分析鉴定。首先采用全扫描-数据依赖型MS/MS（Full MS/dd MS²）采集模式对样品中的肽段信息进行非靶向采集,依次与Swiss-Prot、蛇亚目（Serpentes）、游蛇科（Colubroidea）、眼镜蛇科（Elapidae）、眼镜蛇亚科（Elapinae）、眼镜蛇属（Naja）蛋白质数据库逐级收缩比对;再筛选符合肽谱匹配度、肽段错误发现率小于1%和特征肽段数目大于等于2的蛋白质,共鉴定到32种蛋白质均来自中华眼镜蛇（Naja atra）,可归属于Naja atra的10个家族,主要为三指毒素、金属蛋白酶、磷脂酶A2等。最后,采用平行反应监测模式选取每种蛋白质的两条特征肽段进行靶向验证,当两条特征肽段均满足“至少75%的y⁺和b⁺离子的Δm/z小于5 ppm”时,方认为鉴定到了样品中的某一蛋白质。最终鉴定出5个样品均含有Naja atra蛇毒。此鉴定方法研究系统、严格,可为蛇毒中毒司法鉴定以及毒药物研究等提供有效的技术支持。     

液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白

利用液相等电聚焦预分离技术结合液相色谱-质谱(LTQ-Orbitrap)联用技术,研究了C57小鼠肝脏的蛋白质表达谱.质谱分析结果采用Max Quant1.4.1.2软件搜索数据库,共鉴定出3474个蛋白(2个以上唯一肽段).用DAVID在线工具、GO分类工具和IPA软件对鉴定蛋白进行生物信息学分析,单独发现832个新蛋白.研究结果表明,通过基于等电点和亲疏水性的两维分离,提高了质谱鉴定蛋白数,可以鉴定出更多低丰度蛋白.     

反相液相色谱-串联质谱法鉴定油菜蜂花粉中的蛋白质及活性肽

基于鸟枪法蛋白质组学分析方法,使用反相液相色谱-串联质谱（RPLC-MS/MS）系统分析油菜蜂花粉蛋白质的胰蛋白酶酶解产物,结合数据库检索,共鉴定到353条肽段。鉴定到的肽段所归属的蛋白质中有239个蛋白质可检索到其分子生物学功能,主要功能为结合活性、酶活性、运输活性、抑制活性等。根据血管紧张素转化酶（ACE）抑制肽活性与多肽构效之间的关系,从鉴定到的肽段中筛选并适当修饰后得到5条可能具有ACE抑制活性的肽段,化学合成肽段后进行了活性验证。结果表明5条肽段均具有良好的活性,其中肽段AELDIVLALF和LAVNLIPFP表现出较高的ACE抑制活性,半数抑制浓度（IC₅₀）分别为（10.65±0.50）μmol/L和（23.66±1.08）μmol/L。该方法速度快,成本低,大大缩短了鉴定周期,达到了高通量筛选生物活性肽的目的。     

Epitope peptides and immunotherapy

Allergic diseases affect atopic individuals, who synthesize specific Immunoglobulins E (IgE) to environmental allergens, usually proteins or glycoproteins. These allergens include grass and tree pollens, indoor allergens such as house dust mites and animal dander, and various foods. Because allergen-specific IgE antibodies are the main effector molecules in the immune response to allergens, many studies have focused on the identification of IgE-binding epitopes (called B cell epitopes), specific and minimum regions of allergen molecules that binds to IgE. Our initial studies have provided evidence that only four to five amino acid residues are enough to comprise an epitope, since pentapeptide QQQPP in wheat glutenin is minimally required for IgE binding. Afterwards, various kinds of B cell epitope structures have been clarified. Such information contributes greatly not only to the elucidation of the etiology of allergy, but also to the development of strategies for the treatment and prevention of allergy. Allergen-specific T cells also play an important role in allergy and are obvious targets for intervention in the disease. Currently, the principle approach is to modify B cell epitopes to prevent IgE binding while preserving T cell epitopes to retain the capacity for immunotherapy. There is mounting evidence that the administration of peptide(s) containing immunodominant T cell epitopes from an allergen can induce T cell nonresponsiveness (immunotherapy). There have been clinical studies of peptide immunotherapy performed, the most promising being for bee venom sensitivity. Clinical trials of immunotherapy for cat allergen peptide have also received attention. An alternative strategy for the generation of an effective but hypoallergenic preparation for immunotherapy is to modify T cell epitope peptides by, for example, single amino acid substitution. In this article, I will present an overview of epitopes related to allergic disease, particularly stress on allergen specific immunotherapy. In addition, our ongoing study of immunotherapy by 'eating' T cell epitope peptides will be described. Eating T cell epitope peptides as food provides a more practical way of inducing tolerance and a challenge to prevent allergy in daily life, as opposed to therapy by ingesting peptides as medicine.     

非对称场流分离系统的构建及其在淀粉颗粒粒径表征中的应用

淀粉颗粒粒径与分子尺寸分别在1~100 μm和20~250 nm之间,是影响淀粉功能特性的重要因素之一。非对称场流分离(AF4)是一种基于样品与外力场相互作用机制的分离技术,已应用于表征淀粉分子尺寸分布。商品化的AF4系统的粒径检测范围为1 nm~10 μm,对于淀粉颗粒粒径表征具有一定的局限性。该文研制了AF4分离系统;考察了其在微米尺度下对红薯、莲子和大米淀粉颗粒粒径表征的性能;采用微米尺寸的聚苯乙烯乳化球(PS)标准样品验证了构建的AF4系统的分离性能。实验结果显示,构建的AF4系统对PS混合样品(粒径2、6、12、20 μm)实现了基线分离,同商品化AF4相比提高了检测上线,具有分离表征淀粉颗粒的潜力。此外,该文研究了载液组成对淀粉颗粒分离表征的影响;通过光学显微镜验证了构建的AF4系统在微米尺度上对淀粉颗粒粒径分布的表征能力。最后,采用商品化的AF4系统串联多角度激光光散射检测器和示差折光检测器对3种淀粉分子进行了分离表征,考察了淀粉的溶解温度对其表征结果的影响。在摩尔质量10 ⁶~10⁸ g/mol范围内,红薯和莲子淀粉的回转半径和水合半径的比值(R_g/R_h)在0.9~1.1之间,大米淀粉的R_g/R_h在1.2~1.4之间。实验结果证明构建的AF4系统是一种快速、准确的淀粉颗粒粒径表征方法,与商品化的AF4系统结合可为研究淀粉尺寸分布与其功能性质之间的关系提供技术支持。     

基于非对称场流分离技术分离表征小米淀粉

基于非对称场流分离技术耦合多角度激光光散射检测器和示差折光检测器,建立了分离表征小米淀粉的方法。研究了进样量、交叉流流速、半衰期（t_1/2）、载液离子强度和pH值对小米淀粉分离效果的影响;考察了该方法的重现性;探究了小米淀粉分子结构。结果表明,在进样体积为50 μL、进样质量浓度为0.50 g/L、交叉流流速为1.2 mL/min、t_1/2=3 min、载液为10 mmol/L pH 7.00 NaNO₃（含3 mmol/L NaN₃）的条件下,小米淀粉分离效果最佳。该方法具有良好的重现性,得到的小米淀粉的回转半径相对标准偏差为3.4%、摩尔质量相对标准偏差为7.0%。     

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.     

非对称流场流分离技术的现状及发展趋势

场流分离是生物分析领域一项成熟的技术,将流体与外场联合作用于待分离物质,利用分析物某些理化参数上的差异进行分离。非对称流场流是其重要的分支之一,所施加的外力场为垂直方向的液流,分离过程于开放型的通道中在某种组成的载液迁移推动下进行,主要根据分析物与垂直施加的第二维液流之间的相互作用完成分离。非对称流场流在蛋白质、蛋白质复合物、衍生纳米级/微米级粒子、亚细胞单元和聚合物等分离中的应用日益广泛,主要归功于其直接应用于生物样品时可进行无损分离,因此生物分析物如蛋白质可以在生物友好型的环境中完成分离而不改变其构型,也无需使用降解载液。分离设备便于保持无菌状态,分析物可在生物友好的环境中维持其自然状态。该文简要描述了场流分离原理并罗列出其在生物分析领域一些卓越的发展和应用。     

非对称场流分离技术在蛋黄浆质低密度脂蛋白粒径表征中的应用

通过自组装的非对称场流分离系统(AF4)与紫外可见光检测器联用分离表征了笼养鸡蛋、柴鸡蛋、鹌鹑蛋和鸭蛋蛋黄浆质中的低密度脂蛋白(LDL)。在近似蛋黄浆质生理条件下,研究了进样量、交叉流流速、膜的类型对AF4蛋黄浆质中LDL分离表征的影响;考察了该方法的精密度。在优化的AF4分析条件下,检测出了笼养鸡蛋、柴鸡蛋、鹌鹑蛋和鸭蛋蛋黄浆质中LDL的水力学粒径分布。LDL的AF4洗脱峰高和峰面积的日内精密度分别为1.3%和1.9%(n=7),日间精密度分别为2.4%和2.3%(n=7)。研究结果表明,该方法可用于分离禽类蛋黄浆质中的LDL,同时能够得到LDL水力学粒径分布。     

Epitope mapping of 7S cashew antigen in complex with antibody by solution‐phase H/D exchange monitored by FT‐ICR mass spectrometry

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution‐phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX‐MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope‐contributing segments. Copyright © 2015 John Wiley & Sons, Ltd.     

Asymmetric flow field‐flow fractionation of liposomes: optimization of fractionation variables

The purpose of this study was to investigate the influence of ionic strength of the carrier liquid, cross flow rate, focus flow rate, and sample load on the retention behavior of liposomes in asymmetric flow field‐flow fractionation (AF4). Two differently prepared samples of large unilamellar vesicles (LUV) were used. Experiments were performed varying the factors systematically and evaluating their effect on both retention behavior of the liposomes and on particle size as obtained from online coupled multi‐angle light scattering (MALS) analysis. The results showed that the focus flow rate had the least influence on the elution of liposomes. Elution of LUV is mainly governed by the chosen cross flow condition and ionic strength of the carrier liquid as well as its sample load. Optimal fractionation and size analysis were achieved using a sample load of about 10 μg, a cross flow gradient from 1.0 to 0.1 mL/min over 35 min and a carrier solution of NaNO₃ with a concentration of 10 mM.     

Effect of asymmetrical flow field-flow fractionation channel geometry on separation efficiency

The separation efficiencies of three different asymmetrical flow field-flow fractionation (AF4) channel designs were evaluated using polystyrene latex standards. Channel breadth was held constant for one channel (rectangular profile), and was reduced either linearly (trapezoidal profile) or exponentially (exponential profile) along the length for the other two. The effective void volumes of the three channel types were designed to be equivalent. Theoretically, under certain flow conditions, the mean channel flow velocity of the exponential channel could be arranged to remain constant along the channel length, thereby improving separation in AF4. Particle separation obtained with the exponential channel was compared with particle separation obtained with the trapezoidal and rectangular channels. We demonstrated that at a certain flow rate condition (outflow/inflow rate = 0.2), the exponential channel design indeed provided better performance with respect to the separation of polystyrene nanoparticles in terms of reducing band broadening. While the trapezoidal channel exhibited a little poorer performance than the exponential, the strongly decreasing mean flow velocity in the rectangular channel resulted in serious band broadening, a delay in retention time, and even failure of larger particles to elute.