Multiplex Fluorescent Zdyes for Differential Glycomic Detection

用于差异糖组检测的多重荧光 Zdyes

• 批准号: 7395128
• 负责人: EDWARD A DRATZ
• 金额: $ 19.42万
• 依托单位: ZDYE, LLC
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-03 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7395128
• 关键词: Acids; Bacterial Infections; Benzophenones; Binding; Biological; Biological Process; Biology; Biomedical Research; Boronic Acids; Brain; Businesses; CCR5 gene; CXCR4 gene; Canis familiaris; Carbohydrates; Carbon; Cell Line; Collaborations; Color; Communication; Complement; Complex; Condition; Coupling; Cross-Linking Reagents; Crosslinker; Depth; Detection; Development; Digestion; Disaccharides; Disease; Doctor of Philosophy; Dyes; Esters; Family; Fluorescence; Fluorescent Dyes; Fucose; Galactosides; Gel; Gene Expression; Global Change; Glucosides; Glycol; Glycoproteins; Goals; Health; High Pressure Liquid Chromatography; Human; Hydrogen; Immune response; Isoelectric Point; Label; Mammalian Cell; Mass Spectrum Analysis; Measures; Mediating; Membrane; Monitor; Monosaccharides; Montana; Normal Horse Serum; Object Attachment; Organic Synthesis; Pattern; Peptides; Pharmacology; Phase; Phosphorylation; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteome; Proteomics; Range; Rattus; Reagent; Recovery; Relative (related person); Research; Role; Sampling; Scanning; Signal Transduction; Small Business Technology Transfer Research; Solubility; Spottings; Standards of Weights and Measures; Stimulus; Technology; Testing; Time; Trypsin; Universities; Vascular Endothelial Cell; Vertebral column; Viral; Water; Work; abstracting; base; benzophenone; boronic acid; cancer cell; cell growth regulation; crosslink; design; enzyme activity; experience; glycosylation; improved; internal control; kidney cell; methyl mannoside; multiplex detection; research study; response; sugar; tool; transcription factor

DESCRIPTION (provided by applicant): Protein posttranslational modifications (PTMs) control much of biology. Glycosylation is one of the most common PTMs and more powerful tools are needed to study the occurrence and relative levels of glycosylation. In this proposal we describe the development of new multicolor fluorescent labeling reagents designed for powerful, differential glycomics analysis. The broad, long-term objectives of this work are to simultaneously monitor global changes in levels of (1) proteins, (2) a variety of post-translational modifications, and (3) enzyme activities--in response to biological variables, with greatly improved sensitivity. To accomplish these objectives we are developing (in a cooperative project between Zdye LLC and Montana State University) a new family of ultrasensitive, highly water soluble fluorescent dyes called Zdyes for multicolor, differential labeling--that do not shift the isoelectric points of Zdye-labeled proteins and tend to enhance recovery of labeled proteins. Most PTMs cause shifts in 2D gel spot positions and thus 2D gels provide global pictures of PTM patterns. Our recent work has focused on overcoming past limitations in 2D gel technology and measuring changes in protein levels with biological stimulation. Specific Aims: (1) We will use ortho hydroxymethyl-substituted arylboronic acids to form covalent boronate esters with 1,2-cis diols or 1,3-diols, which are present on all carbohydrates. The ortho hydroxymethyl substituent enhances the binding constants with glycopyranoses about one hundred fold. A benzophenone group will be attached to the arylboronic acid and irradiated to trap the reversible boronate complex. The irradiated benzophenone is designed to abstract a hydrogen atom from the carbohydrate backbone and form a carbon-carbon bond between the carbohydrate and the carbonyl carbon of the benzophenone. Excited benzophenone reacts very poorly with water and if it does not find a C-H to abstract it returns to the ground state to be excited again. Binding and photo-crosslinking will be characterized with a variety of mono and disaccharides. (2) Fluorescent Zdyes will be tethered to the enhanced phenyl boronic acid-benzophenone sugar linker and characterized for crosslinking to a variety of sugars. (3) Glycoprotein standards will be crosslinked with two different-colored Zdye Enhanced Sugar-linkers (Zdye ES-linkers) and conditions optimized for differential labeling and analysis with 1D and 2D gels. (4) Complex protein mixtures from mammalian cell lines expressing human glycoproteins and rat brain samples will be labeled with different colored Zdye ES-linkers and Zdye protein linkers and analyzed on 2D gels to quantify the relative amounts of glycosylation under different biological conditions. Dr. Edward Dratz, PI, is an expert in proteomics, Dr. Paul Grieco, co-PI, is a leader in total organic synthesis, Dr. Mary Cloninger, co-PI, is experienced in carbohydrates and glycoproteins, and Dr. Don Thorne, CEO Zdye LLC has a PhD in pharmacology and expertise in glycoprotein research and business development: a highly synergistic team to develop new glycomics tools. Glycoproteins serve crucial roles in cellular communication and mediate many mechanisms in health and disease. For example, carbohydrate post-translational protein modifications are central to viral and bacterial infection, for metastatic spread of cancer cells, for controlling cellular differentiation, for mounting immune responses, and are involved in modulating transcription factor activity - that control gene expression, in an analogous manner to reversible phosphorylation. Tools to study glycoproteins, however, lag in development at this time and we describe the development of new fluorescence detection technology to determine the relative amounts of glycoproteins in samples exposed to different biological variables, which promise significant advantages for biomedical research.

描述（由申请人提供）：蛋白质翻译后修饰（PTM）控制着大部分生物学。糖基化是最常见的 PTM 之一，需要更强大的工具来研究糖基化的发生和相对水平。在本提案中，我们描述了专为强大的差异糖组学分析而设计的新型多色荧光标记试剂的开发。这项工作的广泛、长期目标是同时监测 (1) 蛋白质、(2) 各种翻译后修饰和 (3) 酶活性水平的全局变化——响应生物变量，大大提高了灵敏度。为了实现这些目标，我们正在开发（在 Zdye LLC 和蒙大拿州立大学之间的一个合作项目中）一种新的超灵敏、高水溶性荧光染料系列，称为 Zdyes，用于多色、差异化标记，不会改变 Zdye 的等电点。标记蛋白质并倾向于提高标记蛋白质的回收率。大多数 PTM 会导致 2D 凝胶点位置发生变化，因此 2D 凝胶可提供 PTM 图案的全局图片。我们最近的工作重点是克服过去二维凝胶技术的局限性，并通过生物刺激测量蛋白质水平的变化。具体目标：（1）我们将使用邻位羟甲基取代的芳基硼酸与所有碳水化合物上存在的1,2-顺式二醇或1,3-二醇形成共价硼酸酯。邻位羟甲基取代基将与吡喃葡萄糖的结合常数增强约一百倍。二苯甲酮基团将连接到芳基硼酸上并进行辐照以捕获可逆硼酸酯络合物。辐照的二苯甲酮旨在从碳水化合物主链中提取氢原子，并在碳水化合物和二苯甲酮的羰基碳之间形成碳-碳键。激发态的二苯甲酮与水的反应非常差，如果它没有找到 C-H 来抽象，它就会回到基态并再次被激发。结合和光交联将以各种单糖和二糖为特征。 (2) 荧光 Zdyes 将与增强型苯基硼酸-二苯甲酮糖连接体连接，并可与多种糖交联。 (3) 糖蛋白标准品将与两种不同颜色的 Zdye 增强型糖连接体（Zdye ES 连接体）交联，并针对 1D 和 2D 凝胶的差异标记和分析进行优化。 (4) 来自表达人糖蛋白的哺乳动物细胞系和大鼠脑样本的复杂蛋白质混合物将用不同颜色的 Zdye ES 连接子和 Zdye 蛋白质连接子标记，并在 2D 凝胶上进行分析，以量化不同生物条件下糖基化的相对量。 Edward Dratz 博士是蛋白质组学方面的专家，Paul Grieco 博士是全有机合成方面的领导者，Mary Cloninger 博士是碳水化合物和糖蛋白领域的专家，Don 博士是Zdye LLC 首席执行官 Thorne 拥有药理学博士学位，并拥有糖蛋白研究和业务开发方面的专业知识：一支高度协同的团队，致力于开发新的糖组学工具。 糖蛋白在细胞通讯中发挥着至关重要的作用，并介导健康和疾病的许多机制。例如，碳水化合物翻译后蛋白修饰对于病毒和细菌感染、癌细胞的转移扩散、控制细胞分化、增强免疫反应至关重要，并且参与调节转录因子活性（控制基因表达）与可逆磷酸化类似的方式。然而，研究糖蛋白的工具目前发展滞后，我们描述了新的荧光检测技术的发展，以确定暴露于不同生物变量的样品中糖蛋白的相对含量，这为生物医学研究带来了显着的优势。