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糖组学研究策略及前沿技术研究进展 总被引:2,自引:0,他引:2
糖组学是继基因组学和蛋白质组学后的新兴研究领域,主要研究聚糖结构与功能.通过与蛋白质组数据库结合,糖捕捉法能系统鉴定糖蛋白和糖基化位点.糖微阵列技术可以对生物个体产生的全部蛋白聚糖结构进行鉴定与表征,提高了聚糖分析通量.而化学选择糖印迹技术简化了聚糖纯化步骤并提高了糖基化分析的灵敏度.双消化并串联柱法通过双酶消化双柱分离,在分析聚糖结构的同时也鉴定蛋白质的序列,并与蛋白质组学研究兼容. 相似文献
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定量蛋白质组研究是蛋白质组研究的热点和难点,而液相色谱质谱技术已经被广泛地应用于蛋白质的定性和定量研究.该研究建立和优化了一种基于液相色谱质谱联用技术的蛋白质组非标记定量方法,并对两种肽段质谱检测计数的归一化算法进行了比较,结果发现ASC法要优于Rsc法.最后,将建立的方法应用于肝癌细胞模型HepG2和HepG2-HBx细胞系的差异蛋白质组表达研究.质谱鉴定结果用聚类分析软件cluster3.0进行分析,最后鉴定出107个重叠蛋白,其中9个蛋白质表达上调(Ratio>1.75),6个蛋白质表达下调(Ratio<0.5),这些蛋白质均与肝癌发生和恶化密切相关.结果表明,该技术操作简单、方便,具有较高的灵敏度和动态范围,利用该方法进行差异蛋白质组研究和发现生物标志物在理论和临床上具有十分重要的意义. 相似文献
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《生命的化学》2015,(5)
随着分离技术、质谱设备与生物信息学的发展,对复杂蛋白质组的深度覆盖研究已不再是遥不可及,对8 000个基因产物的规模化鉴定已经趋于常规化。然而,更深一步的蛋白质组覆盖,就遇到了低丰度蛋白质分离、鉴定等更高的挑战。因此,针对低丰度蛋白质鉴定方法与相关分离分析技术的发展,本文综述了改善低丰度蛋白质鉴定效率的三种方法的最新进展,包括蛋白质的高分辨分离与质谱分析策略、低丰度蛋白质的选择性富集方法和高丰度蛋白质的消减策略等。针对生物体系蛋白质组成的深度鉴定研究,逐步用于探索生命体在不同条件下蛋白质组的表达与修饰的动态变化,并最终发现具备重要生理与病理功能的调控性蛋白质、寻找候选药物靶标。 相似文献
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高分辨率质谱技术的快速发展使得"自顶向下"的蛋白质组学(top-down proteomics)研究逐渐成熟起来.在完整蛋白质水平上研究蛋白质组可以提供更精准、更丰富的生物学信息,特别是对于蛋白质上发生了多种关联性的翻译后修饰的情况.另外,由于基因突变、RNA可变剪接和大量蛋白质翻译后修饰的存在,同一个基因往往最终会产生多个"蛋白质变体"(proteoform),而要准确地鉴定这些蛋白质变体,也离不开"自顶向下"的蛋白质组学.在蛋白质水平上的分离技术、质谱技术与生物信息学技术是完整蛋白质鉴定最关键的三项技术.高效的分离技术是实现规模化蛋白质变体鉴定的前提,有效的质谱碎裂是提供可靠鉴定的核心,而快速准确的质谱鉴定算法则是数据分析效率的保障.本文对这三项技术进行了详细总结,重点集中在生物信息学相关技术上,包括对完整蛋白质的质谱数据预处理、数据库搜索鉴定以及翻译后修饰定位等几个计算问题的讨论. 相似文献
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蛋白质组中蛋白质鉴定技术的研究近况 总被引:8,自引:0,他引:8
蛋白质组学的核心内容之一就是蛋白质的鉴定,基于双向凝胶电泳的图象分析技术可以对组织细胞蛋白质表达的量、表观分子量和等电点等特性进行初步的鉴定,但是对于蛋白质的结构和功能必须借助其它技术手段。目前逐渐形成了以生物质谱为核心的鉴定技术,蛋白质微测和氨基酸组成分析在表达模型分析中也有应用。关于蛋白质组功能模式研究目前可用的方法有酵母双杂交、噬菌体展示、生物传感芯片质谱、蛋白质工程中的定点突变技术等。这些技术对推动蛋白质组学的发展起了一定作用,但是单一技术通常不能确切的鉴定某一蛋白质,常需联合应用几种技术才能准确的鉴定蛋白质。 相似文献
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蛋白质点阵/芯片技术的新进展 总被引:6,自引:0,他引:6
蛋白质点阵/芯片技术是分子生物学技术的重要进展,在功能蛋白质组研究方面具有广阔的潜在应用价值.目前发展起来的印迹蛋白微阵列、分子扫描技术和传感器生物芯片质谱,将应用于药靶检测、疾病诊断、蛋白质结构鉴定和/或蛋白质之间的相互作用分析等方面,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域新的研究工具. 相似文献
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当前,基于生物质谱进行蛋白质鉴定的技术已经成为蛋白质组学研究的支撑技术之一.产生的数据主要使用数据库搜索的方法进行处理,这种方法的一大缺陷是不能鉴定数据库中未包含的蛋白质,因此如何充分利用质谱数据对蛋白质组研究的意义很大,而新蛋白质鉴定更是其中一个重要的内容.新蛋白质鉴定是蛋白质鉴定的一个方面,新蛋白质的定义按照序列和功能的已知程度分为3个层次;以蛋白质鉴定的方法为基础,目前新蛋白质鉴定的方法可分为denovo测序和相似序列搜索结合的方法以及搜索EST、基因组等核酸数据库的方法2大类;两者各有利弊.存在各自的问题和相应处理的策略.不同的研究者可以根据具体目的应用和发展不同的鉴定方法,同时新蛋白质的鉴定也将随着蛋白质组学研究的发展而更加完善. 相似文献
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Birgit An der Lan James L. Cowell Don G. Burstyn Charles R. Manclark Andreas Chrambach 《Molecular and cellular biochemistry》1986,70(1):31-55
Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S2 (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S2, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(10):1646-1649
The replication region of the 2.77-kilobases (kb) plasmid pSTl from Streptococcus thermophilus No. 29 was identified. Deletion derivatives of pSTl were introduced into plasmid-free S. thermophilus No. 29 and examined for their ability to replicate autonomously. The nucleotide sequence had one open reading frame encoded for a 315 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacterial plasmids strongly suggest that the deduced protein (RepS) encoded by pSTl has a replicative role. pSTl also contains a DNA sequence similar to the origin nick sequences of pLPl or pLAB1000, which initiate plasmid replication at the plus origin. In a maxicell system, E. coli CSR603 carrying pSTUC4 produced a protein that was considered to correspond to the product of the RepS gene. These results strongly suggest that pSTl is replicated following a rolling-circle mechanism via single-stranded DNA intermediates. 相似文献
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An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex. 相似文献
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An agarose gel resolving a wide range of DNA fragment lengths 总被引:3,自引:0,他引:3
To resolve DNA fragments ranging from several kilobases to some tens of base pairs in length, an agarose slab gel of steadily increasing thickness has been designed. During electrophoresis a gradient of decreasing electric-field strength is generated throughout the gel from the cathode end to the anode end. Shorter fragments which migrate further are decelerated, resulting in an increased linearity of the relationship between mobility and molecular weight. 相似文献
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Summary Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells
cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate
the removal of cells. The surfaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning
electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most
agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of
the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [3H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests.
The work has been supported by the Swedish Cancer Society and the Swedish Natural Science Research Council. 相似文献
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Electrophoresis in the presence of Coomassie brilliant blue R-250 stains polyacrylamide gels during protein fractionation 总被引:1,自引:0,他引:1
A method of staining polyacrylamide gels in which the dye is electrophoresed together with the sample is proposed. The method cuts short and simplifies the conventional electrophoresis procedure by eliminating the separate poststaining step. In the gels run in the presence of sodium dodecyl sulfate, the method produces protein staining patterns which are quantitatively identical to the ones obtained by conventional staining procedure. Additional advantages of the method are easy control over the degree of staining and homogenous staining independent of the gel thickness and concentration of the dye. 相似文献
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A method for the preparation of uniform gel-disks for enzyme and cell immobilisation, as well as for characterisation of gel mechanical stability, is described. The apparatus comprises a stainless steel base unit and glass parallel plates, designed to permit easy and fast production of multiple homogeneous gel sheets of variable thickness. 相似文献
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蛋白免疫印迹法同时检测大、小分子蛋白的实验条件改进 总被引:1,自引:0,他引:1
目的:蛋白免疫印迹法是现代生物医学研究中广泛应用于蛋白定性和半定量分析的实验技术。然而,常规采用传统单一浓度凝胶的蛋白免疫印迹法在应用过程中仍有不足之处,如不能同时检测分子量很大和很小的蛋白,因而有必要探索一种增大凝胶有效分离范围的检测方法。本文提出采用组合凝胶来实现更大范围分子量蛋白的同时检测。方法:比较双浓度的组合凝胶与单一浓度凝胶的分离范围以及分析采用组合凝胶,蛋白免疫印迹法对大、小分子蛋白的检测效果。结果:12%/7.5%组合凝胶和15%/7.5%组合凝胶的分离范围显著大于相应的单一浓度凝胶。通过12%/7.5%组合凝胶,蛋白免疫印迹法同时检测到15-300 k Da范围内的大、小分子蛋白。结论:组合凝胶有助于蛋白免疫印迹法对分子量相差很大的蛋白进行同时检测分析,具有很强的实用性。 相似文献
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A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins. 相似文献