HistoneScan™: a multiplex immunoassay for histone epigenetic profiling

摘要 基因活性由组蛋白控制，组蛋白与DNA分子相互连接，形成浓缩的染色质 结构.因此，这些组蛋白的变化与失调的生物过程和疾病有关。 到目前为止，DNA技术缺乏精通检测两个或更多个组蛋白修饰的生化测试。 相同的分子。组蛋白特异性抗体的使用可检测有限数量的组蛋白 不能有效地拾取修改并且因此不能有效地拾取复杂的修改模式。发生交叉反应 在相似的蛋白质序列内是这些当前测定的另一个限制。 为了提高复杂的人类组蛋白修饰的检测、鉴定和定量，我们 建议开发一种多重分析测定HistoneScan™。HistoneScan™技术涉及 通过取样初始产生包含感兴趣的组蛋白修饰的组蛋白片段 消化.然后，通过一种技术，将抗体固定到特定的珠子上， 已知为单珠免疫亲和捕获;并且对于一种抗体并入几个珠允许 测定重现性。然后将珠粒对齐到BAMS™载玻片上，以使得能够从其上提取肽。 用于通过质谱分析的相应珠;通过该数据分析确定 特定组蛋白修饰组合。 在这个研究项目中，我们建议开发抗体珠探针，用于捕获多达100个修饰 在人组蛋白H1、H2 A、H2 B、H3和H4中，并开发组蛋白提取和消化 用于优化HistoneScan™工作流程的技术。此外，我们的目标是通过发展加强数据报告， 分析软件HistoneView™，以及HistoneView™作为筛选技术的验证， 用于染色质分析应用。该技术克服了当前测定的主要缺点，即， 蛋白质印迹;设计用于仅测量单一蛋白质变化和变化，以揭示共同发生的 以高通量和具有成本效益的直接方法进行组蛋白修饰。BAMS™的额外使用 这项技术还将允许检测组蛋白修饰， 生物标志物，如组蛋白修饰酶，从而开辟了个性化医疗的道路。

ABSTRACT Gene activity is controlled by histone proteins that interlink with DNA molecules to form condensed chromatin structures. Changes in these histones are thus associated with dysregulated biological processes and disease. To date, DNA technologies lack biochemical tests proficient in detecting two or more histone modifications within the same molecule. The use of histone-specific antibodies confers detection of a limited number of histone modifications and thus complex modification patterns are not picked up effectively. Cross-reactivity occurring within similar protein sequences is an additional limitation of these current assays. In aims of enhancing detection, identification and quantification of complex human histone modifications, we propose the development of a multiplex analytical assay HistoneScan™. HistoneScan™ technology involves initial generation of histone fragments that comprise the histone modification of interest by means of sample digestion. Antibodies immobilized to specific beads are then used to capture modified proteins via a technique known as single bead immunoaffinity capture; and incorporation of several beads for one antibody allows for assay reproducibility. Beads are then aligned onto a BAMS™ slide to enable peptide extraction from their respective beads for analysis by mass spectrometry; by which data analysis determines relative proportions of specific histone modification combinations. In this research project, we propose to develop antibody-bead probes for capture of up to 100 modifications within human histone proteins H1, H2A, H2B, H3 and H4 and to develop a histone extraction and digestion technique for optimized HistoneScan™ workflow. Moreover, we aim to enhance data reporting by development of an analytical software, HistoneView™, and the validation of HistoneView™ as a screening technique to be used for chromatin profiling applications. This technique overrides the main disadvantage of current assays, i.e., Western blots; that are designed to only measure single protein changes and shifts to revealing co-occurring histone modifications in a high-throughput and cost-effective direct approach. The additional use of BAMS™ technology will also allow for the detection of histone modifications that typically serve as useful disease biomarkers, such as histone-modifying enzymes and thus pioneer the way forward in personalized medicine.