Multiplex Fluorescent Zdyes for Differential Glycomic Detection

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。在这个建议中，我们描述了新的荧光标记试剂的开发，设计用于强大的，差异糖组学分析。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）酶活性水平的全球变化-响应生物变量，灵敏度大大提高。为了实现这些目标，我们正在开发（在Zdye LLC和蒙大拿州州立大学之间的合作项目中）一种新的超灵敏度、高水溶性荧光染料家族，称为Zdyes，用于多色、差异化标记-不会改变Zdye标记蛋白质的等电点，并倾向于增强标记蛋白质的回收。大多数PTM引起2D凝胶斑点位置的偏移，因此2D凝胶提供PTM图案的全局图片。我们最近的工作重点是克服2D凝胶技术过去的局限性，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的芳基硼酸与存在于所有碳水化合物上的1，2-顺式二醇或1，3-二醇形成共价硼酸酯。邻位羟甲基取代基增强了与吡喃葡萄糖的结合常数约100倍。将二苯甲酮基团连接到芳基硼酸上并照射以捕获可逆硼酸酯络合物。经辐照的二苯甲酮被设计成从碳水化合物主链上夺取氢原子，并在碳水化合物和二苯甲酮的羰基碳之间形成碳-碳键。激发的二苯甲酮与水的反应非常差，如果它没有找到C-H来提取，它会回到基态再次被激发。结合和光交联将用各种单糖和二糖表征。(2)荧光Z染料将被拴系到增强的苯基硼酸-二苯甲酮糖连接体上，并被表征为与各种糖交联。(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，并优化条件以进行差异标记和1D和2D凝胶分析。(4)将用不同颜色的Zdye ES-接头和Zdye蛋白接头标记来自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，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量。Edward Dratz博士，PI，是蛋白质组学的专家，Paul Grieco博士，联合PI，是全有机合成的领导者，玛丽Cloninger博士，联合PI，在碳水化合物和糖蛋白方面经验丰富，Don Thorne博士，Zdye LLC首席执行官，拥有药理学博士学位和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。 糖蛋白在细胞通讯中起着至关重要的作用，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质修饰对于病毒和细菌感染、癌细胞的转移性扩散、控制细胞分化、增加免疫应答是重要的，并且参与调节转录因子活性-其以与可逆磷酸化类似的方式控制基因表达。然而，研究糖蛋白的工具，在这个时候，滞后的发展，我们描述了新的荧光检测技术的发展，以确定暴露于不同的生物变量，这对生物医学研究的承诺显着优势的样品中的糖蛋白的相对量。

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.