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浏览:- 发布日期:2017-03-20 09:39:54【 】

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analyses: dna, rna and protein

protein analysis

目前已使用标记、蛋白芯片、等方法对蛋白质进行类型鉴定和定量分析()。在二维电泳(two-dimensional electrophoresis,2de)中,蛋白质根据其电荷和分子量分离,随着技术的发展,这种方法已得到显著改善[]。尽管2de早在上世纪70年代就已经开始使用,甚至早于蛋白组学的命名,它仍然在许多实验室中占有十分重要的位置,尤其是它确实对翻译后修饰的分析特别有用[]。通过western blotting从复杂的蛋白质混合物中鉴定特定蛋白时,蛋白质需按其大小分离,然后转移到固体支撑物上,随后使用适当的一级和二级抗体标记靶蛋白[]。

two-dimensional electrophoresis 2d-page used in analysis of proteomes.

进行蛋白质测定需要固体表面(例如显微镜载玻片、膜、珠或微量滴定板以放置蛋白质)、具有多种功能的涂层(以固定蛋白质并防止其变性)、以及亲水环境(用于发生结合反应);其中,使用薄膜技术将涂层施加到载体表面,例如物理气相沉积和化学气相沉积[]。

蛋白质芯片的机制与免疫染色相同。 它利用受体与抗原或细胞表面标记之间的特异性结合。 下图显示了可以使用蛋白质芯片的几个实例。

types of protein microarray.

目前使用的蛋白芯片可分为三类:analytical microarrays(也称作capture arrays或antibody arrays)、functional protein microarrays、reverse-phase protein microarrays(rppa)[]。使用荧光标记(fluorescence labelling)的蛋白芯片是最常见的,其灵敏度高、应用广泛,且与操作简便的激光芯片扫描仪兼容。无需标记(label-free)的检测方法,如表面等离子体共振(surface plasmon resonance )[]、碳纳米管(carbon nanotubes)、碳纳米线传感器(carbon nanowire sensors)和微机电悬臂系统(microelectromechanical system cantilevers)[],这些技术使高通量蛋白质相互作用检测的发展前景更加广阔。

samples of antibody microarray creations and detections.

application of functional protein microarrays in large-scale projects.

outline of reverse-phase protein lysate microarray protocol for quantitative protein expression analysis.

由于蛋白质对微环境变化高度敏感,长时间保持蛋白芯片的稳定状态是非常有难度的。原位技术(in situtechniques)涉及利用无细胞蛋白质表达系统的dna直接在芯片上合成蛋白质,例如dna array to protein array(dapa)[]、protein in situarray(pisa)[]、nucleic acid programmable protein array(nappa)[]等。在拟南芥中已利用串联亲和纯化技术产生17400个orf,用于开发用于大规模蛋白质分析和重组拟南芥蛋白生产的平台。利用纯化的融合蛋白印迹,现已生产出高密度拟南芥蛋白芯片,用于蛋白互作分析[,]和蛋白鉴定[]。对translated orfs进行平行分析可以发现其互作蛋白[]。

schematic diagram of dapa.

diagram of pisa.

diagram of nappa.

直接确定蛋白质氨基酸序列的方法(protein-seq)主要有两种,即质谱法(mass spectrometry,ms)和edman degradation。得益于其高效与高灵敏度,ms不仅是研究蛋白质一级结构的主要技术,也成为蛋白质组学研究的中心技术。蛋白质量可以通过将稳定的同位素或质量标签添加到不同的样品中来确定,从而通过质量的特定增加来鉴定等价肽(或肽片段)。当蛋白质和肽通过选择标签标记时,例如isotope-coded affinity tags[]、绝对和相对定量isobaric tags[],可以测定有限数量的蛋白质。利用非选择性标签的,例如isotope-coded protein label[]、mass-coded abundance tagging(mcat)[],可以实现大规模肽和定量。如果样品在仍然具有代谢活性(在细胞培养物中用氨基酸稳定同位素标记,silac)时被标记,则可以量化数千种蛋白质的动态体内特征[]。通过结合silac和itraq,可以实现18重同位素标记,且已有允许测试六种不同蛋白质样品的两阶段稳定同位素标记策略被开发出来[]。

mass spectrometry protocol.

mechanism of edman degradation.

reference

[1] grishkevich v, yanai i. the genomic determinants of genotype× environment interactions in gene expression[j]. trends in genetics, 2013, 29(8): 479-487.

[2] ramirez‐gonzalez r h, segovia v, bird n, et al. rna‐seq bulked segregant analysis enables the identification of high‐resolution genetic markers for breeding in hexaploid wheat[j]. plant biotechnology journal, 2015, 13(5): 613-624.

[3] michelmore r w, paran i, kesseli r v. identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations[j]. proceedings of the national academy of sciences, 1991, 88(21): 9828-9832.

[4] ghazvini h, hiebert c w, thomas j b, et al. development of a multiple bulked segregant analysis (mbsa) method used to locate a new stem rust resistance gene (sr54) in the winter wheat cultivar norin 40[j]. theoretical and applied genetics, 2013, 126(2): 443-449.

[5] zhu h, snyder m. protein chip technology[j]. current opinion in chemical biology, 2003, 7(1): 55-63.

[6] kover p x, valdar w, trakalo j, et al. a multiparent advanced generation inter-cross to fine-map quantitative traits in arabidopsis thaliana[j]. plos genet, 2009, 5(7): e1000551.

[7] zhang j, kruss s, hilmer a j, et al. a rapid, direct, quantitative, and label‐free detector of cardiac biomarker troponin t using near‐infrared fluorescent single‐walled carbon nanotube sensors[j]. advanced healthcare materials, 2014, 3(3): 412-423.

[8] he m, stoevesandt o, taussig m j. in situ synthesis of protein arrays[j]. current opinion in biotechnology, 2008, 19(1): 4-9.

[9] he j, zhao x, laroche a, et al. genotyping-by-sequencing (gbs), an ultimate marker-assisted selection (mas) tool to accelerate plant breeding[j]. frontiers in plant science, 2014, 5: 484.

[10] nagano a j, sato y, mihara m, et al. deciphering and prediction of tranome dynamics under fluctuating field conditions[j]. cell, 2012, 151(6): 1358-1369.

[11] popescu s c, popescu g v, bachan s, et al. mapk target networks in arabidopsis thaliana revealed using functional protein microarrays[j]. genes & development, 2009, 23(1): 80-92.

[12] lee y p, cho y, kim s. a high-resolution linkage map of the rfd1, a restorer-of-fertility locus for cytoplasmic male sterility in radish (raphanus sativus l.) produced by a combination of bulked segregant analysis and rna-seq[j]. theoretical and applied genetics, 2014, 127(10): 2243-2252.

[13] rabilloud t, chevallet m, luche s, et al. two-dimensional gel electrophoresis in proteomics: past, present and future[j]. journal of proteomics, 2010, 73(11): 2064-2077.

[14] lee h y, bowen c h, popescu g v, et al. arabidopsis rtnlb1 and rtnlb2 reticulon-like proteins regulate intracellular trafficking and activity of the fls2 immune receptor[j]. the plant cell, 2011, 23(9): 3374-3391.

[15] hall d a, ptacek j, snyder m. protein microarray technology[j]. mechanisms of ageing and development, 2007, 128(1): 161-167.

[16] routaboul j m, dubos c, beck g, et al. metabolite profiling and quantitative genetics of natural variation for flavonoids in arabidopsis[j]. journal of experimental botany, 2012, 63(10): 3749-3764.

[17] kim s y, li y, guo y, et al. design of association studies with pooled or un‐pooled next‐generation sequencing data[j]. genetic epidemiology, 2010, 34(5): 479-491.

[18] chan e k f, rowe h c, corwin j a, et al. combining genome-wide association mapping and tranional networks to identify novel genes controlling glucosinolates in arabidopsis thaliana[j]. plos biol, 2011, 9(8): e1001125.

[19] deng z, zhang x, tang w, et al. a proteomics study of brassinosteroid response in arabidopsis[j]. molecular & cellular proteomics, 2007, 6(12): 2058-2071.

[20] wang b, du q, yang x, et al. identification and characterization of nuclear genes involved in photosynthesis in populus[j]. bmc plant biology, 2014, 14(1): 1.

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