Multiplex Fluorescent Zdyes for Differential Glycomic Detection

描述（由申请人提供）：蛋白质翻译后修饰（PTMs）控制着许多生物学。糖基化是最常见的PTMs之一，需要更强大的工具来研究糖基化的发生和相对水平。在这个建议中，我们描述了新的多色荧光标记试剂设计的强大的，差异糖组学分析的发展。这项工作的广泛、长期目标是同时监测(1)蛋白质水平的全球变化，(2)各种翻译后修饰，(3)酶活性——响应于生物变量，灵敏度大大提高。为了实现这些目标，我们正在开发（在Zdye LLC和蒙大拿州立大学的合作项目中）一个新的超灵敏、高水溶性荧光染料家族，称为多色、差分标记的Zdye，它不会改变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引起二维凝胶点位置的变化，因此二维凝胶提供了PTM模式的全局图像。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。具体目的：(1)我们将使用邻羟基甲基取代的芳基硼酸与所有碳水化合物中存在的1,2-顺式二醇或1,3-二醇形成共价硼酸酯。邻羟基甲基取代基使其与甘氨糖的结合常数提高了约100倍。一个二苯甲酮基团将连接到芳基硼酸上并照射以捕获可逆的硼酸配合物。辐照二苯甲酮被设计成从碳水化合物主链中提取一个氢原子，并在碳水化合物和二苯甲酮的羰基碳之间形成碳碳键。受激的二苯甲酮与水的反应很差如果它找不到一个碳氢化合物来抽离它就会回到基态再次被激发。结合和光交联将以各种单糖和双糖为特征。(2)荧光染料将连接到增强的苯硼酸-二苯甲酮糖连接剂上，并表征为与多种糖交联。(3)糖蛋白标准品将与两种不同颜色的Zdye Enhanced Sugar-linkers （Zdye ES-linkers）交联，并优化条件，用于1D和2D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样品的复杂蛋白混合物用不同颜色的Zdye ES-linkers和Zdye protein linkers进行标记，并在2D凝胶上进行分析，量化不同生物学条件下糖基化的相对量。爱德华·德拉茨博士是蛋白质组学专家，保罗·格里科博士是全有机合成领域的领导者，玛丽·克罗宁格博士是碳水化合物和糖蛋白领域的资深专家，而唐·索恩博士是Zdye LLC的首席执行官，拥有药理学博士学位，糖蛋白研究和业务开发方面的专业知识：一个高度协同的团队，开发新的糖组学工具。

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.