Photocleavable Bead Technology for Glycomics

蛋白质的翻译后修饰（PTMs）在多种细胞过程中发挥核心作用，包括蛋白质折叠、靶向、信号转导、免疫反应、粘附、运动和蛋白质降解。超过300种不同类型的ptm已经被发现，并且在大约80%的蛋白质中被发现，部分解释了与基因组相比，蛋白质组要大得多的原因。越来越多的人认识到，表征这些ptm及其如何调节蛋白质功能的重要性，对于了解疾病的分子基础、发现新的诊断/预后生物标志物、开发新的药物疗法，甚至了解不同病毒与细胞受体的相互作用，都至关重要。然而，在开发能够检测和分析PTMs的有效技术方面存在许多挑战，PTMs可能非常复杂，特别是在蛋白质糖基化的情况下。正如该资助申请中所述，“碳水化合物结构的分离、分析定量和详细表征策略是核心挑战”。近年来，利用糖基化肽（O-PTMs）和光生成碳水化合物阵列等聚糖微阵列筛选糖基化PTMs的研究取得了进展。然而，蛋白质微阵列技术的局限性，包括相对较低的密度，特别是在排列大型蛋白质/肽库时，较差的可重复性和较差的分析动力学，使得这种方法不太理想。此外，与质谱法不同，质谱法通常用于分析肽和蛋白质的糖基化，微阵列不能提供这样的信息。大型组合糖肽珠库提供了微阵列的替代方案，但通常使用“平移”方法来测量与库的相互作用，需要手动“挑选”大型单珠，以便随后通过质谱逐一分析。在第一阶段，我们将开发一种新的糖组学方法，该方法结合了质谱法和AmberGen开发的光可切割连接技术的优势。在一个例子中，一个光可切割的糖肽头库将被合成并随机纳入一个高密度的微孔板，以形成一个阵列。正如在初步实验中所证明的那样，这种方法可以在头阵列中以高灵敏度和空间分辨率快速测量潜在的数百万种不同的“诱饵”糖肽，从而实现糖结合蛋白（GBPs）、糖苷酶/糖转移酶、激酶和药物等相互作用生物分子的作用。在第二种非阵列的例子中，光可切割的糖肽头文库用含有特定“猎物”类型（例如血清自身抗体）的生物标本处理。然后使用“照片释放和重新捕获”工作流程快速地将糖肽-猎物复合物富集到非常高的纯度。接下来是常规的基于质谱的蛋白质组学分析，以鉴定相互作用的诱饵糖肽，从而快速鉴定用于疾病诊断和治疗的潜在生物标志物。第三种方法建立在最近报道的使用AmberGen光可切割连接剂的基础上，用于鉴定细胞、组织和其他生物标本中的o -连接β - n -乙酰氨基葡萄糖（O-GlcNAc）蛋白修饰。这些修饰的重要性已经与磷酸化进行了比较，然而我们准确检测和表征它们的能力刚刚出现了令人兴奋的新方法。在这里，我们将改进这些方法，使用专有的光可切割同位素编码亲和标记试剂（PC-ICAT）进行定量糖蛋白组学，以确定O-GlcNAc模式如何变化，例如在正常和患病状态下。为了加速该项目产生的方法和产品的商业化，我们将在第一阶段和第二阶段与世界领先的MALDI-MS仪器供应商Bruker Daltonics （Billerica， MA）密切合作（见业务发展副总裁Gary Kruppa博士的信）。此外，我们将与丹麦哥本哈根大学糖组学中心的Ola Blixt博士合作，他是糖肽库合成方法的开发人员，以及波士顿大学生物医学质谱中心主任、人类蛋白质组组织主席、生物化学、生物物理和化学教授Cathy Costello博士合作，他是公认的基于质谱的糖组学技术专家（见两位博士的合作信函）。Blixt和Costello)。

DESCRIPTION: Post-translational modifications of proteins (PTMs) play a central role in diverse cellular processes including protein folding, targeting, signal transduction, immune response, adherence, motility and protein degradation. Over 300 different types of PTMs are already known and are found in an estimated 80% of all proteins, accounting in part for the vastly larger proteome compared to the genome. Increasingly, the importance of characterizing these PTMs and how they modulate protein function is being recognized as crucial to understanding the molecular basis for disease, as well as to the discovery of new diagnostic/prognostic biomarkers, development of new drug therapies and even understanding the interaction of different viruses with cell receptors. However, many challenges exist in developing effective techniques that can detect and analyze PTMs which can be highly complex, especially in the case of glycosylation of proteins. As stated in this grant solicitation "Strategies for separation, profiling quantitation and detailed characterization of carbohydrate structures are central challenges". Recently, progress has been made towards screening glycomic PTMs using glycan microarrays including arrays of O-glycosylated peptides (O-PTMs) and photo-generated carbohydrate arrays. However, limitations in protein microarray technology, including relatively low density especially when arraying large protein/peptide libraries, poor reproducibility, and poor assay kinetics, make this approach less than ideal. In addition, unlike mass spectrometry, which is conventionally used to analyze glycosylation of peptides and proteins, microarrays do not provide such information. Large combinatorial bead-libraries of glycopeptides offer an alternative to microarrays, but normally utilize "panning" methods to measure interactions with the library, requiring manual "picking" of large, single beads for subsequent one-by-one analysis by mass spectrometry. During Phase I we will develop a new approach to glycomics which combines the advantages of mass spectrometry and photocleavable linker technology developed by AmberGen. In one example, a photocleavable glycopeptide bead library will be synthesized and randomly incorporated into a high-density Pico-well plate to form an array. As demonstrated in preliminary experiments, this approach allows the effects of interacting biomolecules such as glycan binding proteins (GBPs), glycosidases/glycotransferases, kinases and drugs to be rapidly measured on potentially millions of different "bait" glycopeptides in the bead-array, with high sensitivity and spatial resolution. In a second, non-array based example, the photocleavable glycopeptide bead library is treated with a biospecimens containing a particular "prey" type of interest (e.g. a serum autoantibody). Glycopeptide-prey complexes are then rapidly photo-enriched to very high purity using a "photo-release and re-capture" workflow. This is followed by conventional mass spectrometry-based proteomic analysis to identify the interacting bait glycopeptides, allowing rapid identification of potential biomarkers for disease diagnosis and treatment. A third approach builds on the recently reported use of AmberGen's photocleavable linkers to identify O-linked beta- N-acetylglucosamine (O-GlcNAc) protein modifications in cells, tissues and other biospecimens. The importance of these modifications has been compared to phosphorylation, yet our ability to accurately detect and characterize them is just now emerging with exciting new methods. Here, we will improve upon these methods by using proprietary photocleavable isotope coded affinity tagging reagents (PC-ICAT) for quantitative glycoproteomics to determine how O-GlcNAc patterns change, e.g. in normal and diseased states. In order to accelerate commercialization of the methods and products resulting from this project we will work closely during Phase I and II with Bruker Daltonics (Billerica, MA), a world-leading provider of MALDI-MS instrumentation (see letter from Dr. Gary Kruppa, V.P. of Business Development). In addition, we will collaborate with Dr. Ola Blixt of the Center for Glycomics, Copenhagen University in Denmark, the developer of robust methods for synthesis of glycopeptide libraries, and Dr. Cathy Costello, Director, Boston University Center for Biomedical Mass Spectrometry, President, Human Proteome Organization, and Professor, Biochemistry, Biophysics and Chemistry who is a recognized expert in mass spectrometry based glycomics techniques (see letters of collaboration from both Drs. Blixt and Costello).