基于DNA模板光交联生物质谱鉴定结合蛋白的研究

发布时间：2018-07-12 11:11

本文选题：液相色谱-质谱 + 适配体　；参考：《天津医科大学》2017年硕士论文

【摘要】：DNA或RNA适配体具有选择性识别病变细胞的能力,在生物标志物的发现和疾病早期诊断方面显示了应用潜力。目前的研究表明:适配体是通过与细胞表面特异性蛋白的结合来实现对细胞的分辨。然而由于适配体与细胞表面靶标蛋白之间的作用力往往很弱,因此精准鉴定适配体的结合蛋白仍然非常困难,已成为瓶颈问题之一,迫切需要发展高灵敏的分析方法。组蛋白翻译后修饰(Histone Post-translational modifications,HPTMs)可以通过招募结合蛋白及复合物调控基因转录。最近研究发现:临近的组蛋白修饰具有协调或拮抗的作用,改变与结合蛋白的作用,从而影响转录。然而多种修饰介导的蛋白作用的表征和检测非常困难,国际上一直缺乏有效的分析手段。针对以上问题,我们提出“发展鉴定结合蛋白的高灵敏分析方法”。本文分别以适配体、修饰多肽为诱饵,构建了基于DNA模板和光交联技术的分子探针,联合生物质谱技术,分别建立了适配体识别蛋白和组蛋白多修饰结合蛋白的高灵敏分析方法。研究内容主要分为以下两个部分:第一部分:基于DNA模板化学和光交联技术,利用核酸适配体的识别特性,以溶菌酶为模型,设计了一种核酸适配体分子探针,用于研究目标蛋白溶菌酶的选择性标记和富集。一方面,探针可将较弱的“核酸-蛋白”分子间作用转化为共价键合,提高了适配体结合蛋白的捕获;另一方面,将交联剂固载在另一条独立的单链DNA上,从而降低了以往交联剂对适配体与结合蛋白专一性识别的干扰。在优化的条件下,探针对溶菌酶显示出高灵敏性和高选择性的标记和富集,实现了复杂背景下以及实际样品中对目标蛋白的标记和富集,并通过质谱进行了鉴定。实验结果显示这一新方法在发现适配体结合蛋白方面具有潜力。第二部分:基于多肽修饰识别、DNA自组装和亲和光交联技术,我们设计了多肽探针,探索多种修饰介导的结合蛋白分析新方法,并以此研究了组蛋白H3T3(苏氨酸)磷酸化/H3K4(赖氨酸)三甲基与蛋白结构域PHD的互作。实验结果表明:H3T3磷酸化抑制了H3K4三甲基化对PHD的识别,从分析化学层面验证了文献报道的发现。我们的研究显示:建立的方法可用于分析多种修饰介导下的结合蛋白,以及修饰间相互作用,在生物医学中具有潜在应用价值。
[Abstract]:DNA or RNA aptamers have the ability to selectively recognize diseased cells, and have shown potential application in the discovery of biomarkers and early diagnosis of diseases. Current studies have shown that aptamers can differentiate cells by binding to cell surface specific proteins. However, due to the weak interaction between aptamers and target proteins on cell surface, it is still very difficult to accurately identify the binding proteins of aptamers, which has become one of the bottleneck problems, and it is urgent to develop highly sensitive analytical methods. Histone Post-translational modifications (HPTMs) regulate gene transcription by recruiting binding proteins and complexes. Recent studies have found that adjacent histone modifications have coordinated or antagonistic effects, altering and binding proteins, thus affecting transcription. However, the characterization and detection of proteins mediated by various modifications are very difficult, and there has been a lack of effective analytical methods in the world. In view of the above problems, we proposed a highly sensitive method for the development and identification of binding proteins. In this paper, molecular probes based on DNA template and photocrosslinking technique were constructed with aptamer and modified polypeptide as bait respectively. A highly sensitive method for the analysis of aptamer recognition protein and histone multi-modified binding protein was established. The main contents are as follows: in the first part, based on DNA template chemistry and photocrosslinking technology, a novel aptamer molecular probe was designed based on the recognition characteristics of aptamer and lysozyme. It is used to study the selective labeling and enrichment of the target protein lysozyme. On the one hand, the probe can convert the weak "nucleic acid-protein" intermolecular interaction into covalent binding, which enhances the capture of aptamer binding proteins; on the other hand, the cross-linking agent is immobilized on another independent single-stranded DNA. Thus, the interference of previous crosslinking agents on the specificity recognition of aptamer and binding protein was reduced. Under the optimized conditions, the probe showed high sensitivity and high selectivity in labeling and enrichment of lysozyme. The target protein was labeled and enriched in complex background and in actual samples, and was identified by mass spectrometry. The results show that this new method has potential in the discovery of aptamer binding proteins. Part two: based on the techniques of polypeptide modification recognition DNA self-assembly and photocrosslinking we designed polypeptide probes and explored a variety of novel methods for the analysis of binding proteins mediated by modification. The interaction between histone H3T3 (threonine) phosphorylated / H3K4 (lysine) trimethyl and protein domain PhD was studied. The results showed that the phosphorylation of H _ 3T _ 3 inhibited the recognition of PhD by H _ 3K _ 4 trimethylation. Our results show that the proposed method can be used for the analysis of binding proteins mediated by various modifications and the interaction between modifications, and has potential application value in biomedicine.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416

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