Photocleavable Bead Technology for Glycomics

产品说明： 蛋白质的翻译后修饰（Posttranslational modifications of proteins，PTM）在蛋白质折叠、靶向、信号转导、免疫应答、粘附、运动和蛋白质降解等多种细胞过程中发挥着重要作用。已知有超过300种不同类型的PTM，并且在估计80%的所有蛋白质中发现，部分原因是与基因组相比，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至关重要。然而，在开发可以检测和分析可能高度复杂的PTM的有效技术方面存在许多挑战，特别是在蛋白质糖基化的情况下。正如在这份资助申请中所述，“分离、分析定量和详细表征碳水化合物结构的策略是核心挑战”。最近，使用聚糖微阵列（包括O-糖基化肽（O-PTM）阵列和光生碳水化合物阵列）筛选糖基PTM已经取得了进展。然而，蛋白质微阵列技术的局限性，包括相对低的密度，特别是当排列大的蛋白质/肽文库时，差的再现性和差的测定动力学，使得这种方法不太理想。此外，与常规用于分析肽和蛋白质的糖基化的质谱法不同，微阵列不提供这样的信息。糖肽的大型组合珠库提供了微阵列的替代方案，但通常利用“淘选”方法来测量与库的相互作用，需要手动“挑选”大的单个珠用于随后通过质谱法进行的逐个分析。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开发的光可裂解接头技术的优势。在一个实例中，将合成光可裂解糖肽珠文库并将其随机掺入高密度皮可孔板中以形成阵列。如在初步实验中所证明的，这种方法允许相互作用的生物分子如聚糖结合蛋白（GBP）、糖苷酶/糖基转移酶、激酶和药物的作用以高灵敏度和空间分辨率对珠阵列中潜在的数百万种不同的“诱饵”糖肽进行快速测量。在第二个非基于阵列的实施例中，用含有特定目标“猎物”类型（例如血清自身抗体）的生物样本处理光可裂解糖肽珠文库。然后使用“光释放和再捕获”工作流程将糖肽-猎物复合物快速光富集至非常高的纯度。随后进行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的光裂解接头来鉴定细胞、组织和其他生物标本中的O-连接的β-N-乙酰葡糖胺（O-GlcNAc）蛋白修饰的基础上。这些修饰的重要性已经与磷酸化进行了比较，但我们准确检测和表征它们的能力现在正在出现令人兴奋的新方法。在这里，我们将通过使用专有的光可裂解同位素编码的亲和标记试剂（PC-ICAT）进行定量糖蛋白组学来改进这些方法，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（马萨诸塞州比勒里卡）密切合作（见业务发展副总裁加里克虏伯博士的来信）。此外，我们还将与丹麦哥本哈根大学糖组学中心的奥拉布利克斯特博士（糖肽库合成的稳健方法的开发者）和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兼生物化学教授凯茜·科斯特洛博士（Cathy Costello）合作，生物物理学和化学，是公认的基于质谱的糖组学技术专家（参见Blixt博士和Costello博士的合作信）。

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).