HistoneScan™: a multiplex immunoassay for histone epigenetic profiling

HistoneScan™：用于组蛋白表观遗传分析的多重免疫分析

• 批准号: 10654855
• 负责人: Vladislav Bergo
• 金额: $ 43.69万
• 依托单位: ADEPTRIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654855
• 关键词: Address; Amino Acid Sequence; Antibodies; Biochemical; Biological; Biological Assay; Biological Markers; Biological Process; Cell Culture Techniques; Cells; ChIP-seq; Chromatin; Chromatin Structure; Companions; Complex; Computer software; Custom; DNA; Data; Data Analyses; Data Reporting; Data Set; Detection; Development; Digestion; Disadvantaged; Disease; Enzyme-Linked Immunosorbent Assay; Enzymes; Epigenetic Process; Generations; Genes; Genetic Transcription; Hela Cells; Histone Deacetylase Inhibitor; Histone H1; Histones; Hour; Human; Immobilization; Immunoassay; Incidence; Lysine; Mass Spectrum Analysis; Measures; Methods; Methylation; Modification; Pattern; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Preparation; Process; Proteins; Proteomics; Protocols documentation; Reagent; Recovery; Regulator Genes; Reporting; Reproducibility; Research; Research Project Grants; Resources; Sampling; Screening procedure; Services; Signal Transduction; Site; Slide; Techniques; Technology; Testing; Time; Validation; Vorinostat; Western Blotting; antibody conjugate; biomarker validation; combinatorial; cost effective; cross reactivity; design; epigenetic profiling; experimental study; histone modification; interest; personalized medicine; precision medicine; product development; screening; synthetic peptide

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.

抽象的 基因活性由与DNA分子相互链接以形成冷凝染色质的组蛋白控制 结构。因此，这些组蛋白的变化与生物学过程和疾病失调有关。 迄今 相同的分子。组蛋白特异性抗体的使用承认检测有限数量的组蛋白 修改并因此无法有效地拾取复杂的修改模式。发生交叉反应性 在相似的蛋白质序列中，是这些当前测定的另一个限制。 为了增强复杂人类Hisstone修改的检测，鉴定和量化，我们 提案开发多重分析测定HampOnescan™。 HampOnescan™技术涉及 通过样品的初始生成组成的组蛋白片段，该片段包含了感兴趣的组蛋白的修饰 消化。然后使用固定在特定珠的抗体通过技术捕获修饰的蛋白质 被称为单珠免疫亲和力捕获；并掺入一种用于一种抗体的珠子允许 测定可重复性。然后将珠子对齐到BAMS™载玻片上，以使肽从其 通过质谱法进行分析的相对珠；数据分析确定相对比例 特定的Hisstone修改组合。 在该研究项目中，我们建议开发抗体珠问题，以捕获多达100个修改 在人类组蛋白H1，H2A，H2B，H3和H4中 优化HampOnescan™工作流程的技术。此外，我们旨在通过开发增强数据报告 分析软件，HastoneView™和HistoneView™作为筛选技术的验证 用于染色质分析应用。该技术超过了当前测定的主要灾难，即 蛋白质印迹；旨在仅测量单个蛋白质的变化和转移向揭示同时发生的 Hisstone修改高通量和具有成本效益的直接方法。 BAMS™的额外使用 技术还将允许检测通常用作有用疾病的组蛋白修饰 生物标志物，例如组蛋白改良酶，从而在个性化医学中开创了前进的道路。