Multiplex Fluorescent Zdyes for Differential Glycomic Detection

描述(申请人提供)：蛋白质翻译后修饰(PTM)控制着生物学的大部分。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。在这项提案中，我们描述了新的多色荧光标记试剂的发展，该试剂设计用于强大的、差别化的糖组分分析。这项工作的广泛和长期目标是同时监测(1)蛋白质、(2)各种翻译后修饰和(3)酶活性水平的全球变化--对生物变量的反应，并大大提高灵敏度。为了实现这些目标，我们正在(在ZdyLLC和蒙大拿州立大学的一个合作项目中)开发一种名为Zdyes的新的超灵敏、高度水溶性的荧光染料系列，用于多色、差异标记--这种染料不会移动Zdye标记的蛋白质的等电点，并倾向于提高标记蛋白质的回收率。大多数PTM会引起2D凝胶点位置的移动，因此2D凝胶提供了PTM图案的全局图像。我们最近的工作重点是克服过去2D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)我们将使用邻羟甲基取代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。邻位羟甲基取代基提高了与糖苷类化合物的结合常数约100倍。二苯甲酮基团将被连接到芳基硼酸上，并被辐射以捕获可逆的硼酸盐络合物。辐照的二苯甲酮被设计成从碳水化合物骨架中提取氢原子，并在碳水化合物和二苯甲酮的羰基碳之间形成碳-碳键。被激发的二苯甲酮与水的反应很差，如果它没有找到一个C-H来抽提，它就会回到基态再次被激发。结合和光交联会以各种单糖和双糖为特征。(2)荧光ZDYS将被连接到增强的苯基硼酸-二苯甲酮糖链上，并被表征为与多种糖交联。(3)糖蛋白标准品将用两种不同颜色的Zdy增强型糖链(ZdyEs-Linker)进行交联，并优化条件，用于一维和二维凝胶的差异标记和分析。(4)将表达人糖蛋白的哺乳动物细胞系和大鼠脑标本的复合蛋白混合物分别标记为不同颜色的ZdyES连接物和Zdy蛋白连接物，并在2D凝胶上进行分析，以定量不同生物条件下糖基化的相对数量。Pi的Edward Dr.Dr.Edward是蛋白质组学专家，Pi的Paul Grieco博士是全有机合成领域的领导者，Pi的联席Mary Cloninger博士在碳水化合物和糖蛋白方面经验丰富，而ZdyLLC的CEO Don Thorne博士拥有药理学博士学位，并在糖蛋白研究和业务开发方面拥有专业知识：一个高度协同的团队，开发新的糖组学工具。 糖蛋白在细胞通讯中起着至关重要的作用，并在健康和疾病中调节许多机制。例如，碳水化合物翻译后蛋白修饰是病毒和细菌感染、癌细胞转移扩散、控制细胞分化、增强免疫反应的核心，并参与调节转录因子的活性--以类似于可逆磷酸化的方式控制基因的表达。然而，目前研究糖蛋白的工具发展滞后，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数量，这将为生物医学研究带来显著的优势。

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.