Photocleavable Bead Technology for Glycomics

描述：蛋白质翻译后修饰(PTM)在蛋白质折叠、靶向、信号转导、免疫反应、黏附、运动和蛋白质降解等多种细胞过程中发挥核心作用。已知300多种不同类型的PTM，估计在所有蛋白质中发现了80%，这在一定程度上解释了与基因组相比，蛋白质组要大得多。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，对于理解疾病的分子基础，以及发现新的诊断/预后生物标志物，开发新的药物疗法，甚至理解不同病毒与细胞受体的相互作用都是至关重要的。然而，在开发有效的技术来检测和分析PTMS方面存在许多挑战，这些技术可能非常复杂，特别是在蛋白质糖基化的情况下。正如在这次赠款征集中所说的，“碳水化合物结构的分离、图谱定量和详细描述的策略是主要的挑战”。最近，利用包括O-糖基化多肽(O-PTM)阵列和光生碳水化合物阵列在内的葡聚糖微阵列筛选糖基化PTM的研究取得了进展。然而，蛋白质微阵列技术的局限性，包括相对低的密度，特别是在排列大型蛋白质/肽文库时，重复性差，以及糟糕的分析动力学，使这种方法不太理想。此外，与传统上用于分析多肽和蛋白质糖基化的质谱学不同，微阵列不提供此类信息。大的糖肽组合珠库提供了微阵列的另一种选择，但通常使用“摇摄”方法来测量与文库的相互作用，需要人工“挑选”大的、单一的珠子，以便随后通过质谱学进行逐个分析。在第一阶段，我们将开发一种新的糖组学方法，它结合了质谱学和AmberGen开发的光解连接子技术的优势。在一个实例中，将合成可光裂解的糖肽珠库并随机结合到高密度皮科孔板中以形成阵列。在初步实验中，这种方法允许相互作用的生物分子，如糖结合蛋白(GBP)、糖苷酶/糖转移酶、激酶和药物的影响，以高灵敏度和空间分辨率快速地在微珠阵列中的潜在的数百万个不同的“诱饵”糖肽上被测量。在基于非阵列的第二个例子中，用包含特定的感兴趣的“猎物”类型(例如血清自身抗体)的生物检验剂处理可光裂解的糖肽珠库。糖肽-猎物复合体随后通过“光释放和重新捕获”的工作流程被快速光浓缩到非常高的纯度。这之后是传统的基于质谱学的蛋白质组分析，以确定相互作用的诱饵糖肽，允许快速识别潜在的生物标记物，用于疾病诊断和治疗。第三种方法建立在最近报道的使用AmberGen的可光裂解连接物来识别细胞、组织和其他生物有机体中O-连接的β-N-乙酰氨基葡萄糖(O-GlcNAc)蛋白质修饰的基础上。这些修饰的重要性已经被比作磷酸化，但我们准确检测和表征它们的能力刚刚出现，出现了令人兴奋的新方法。在这里，我们将改进这些方法，使用专利的光切割同位素编码的亲和标记试剂(PC-ICAT)来定量糖蛋白组学，以确定O-GlcNAc模式是如何变化的，例如在正常和疾病状态下。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(马萨诸塞州比勒里卡)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链中心的Ola Blixt博士和波士顿大学生物医学质谱学中心主任、人类蛋白质组组织主席Cathy Costello博士合作，Ola Blixt博士是糖肽库合成稳健方法的开发商，Cathy Costello博士是公认的基于质谱学的糖组技术专家(见Blixt博士和Costello博士的合作信函)。

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).