Photocleavable Bead Technology for Glycomics

用于糖组学的光裂解珠技术

• 批准号: 8455590
• 负责人: Mark Lim
• 金额: $ 34.92万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-28 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455590
• 关键词: Accounting; Acetylglucosamine; Adherence; Alkynes; Autoantibodies; Binding; Binding Proteins; Biochemistry; Biological Assay; Biological Markers; Biophysics; Boston; Businesses; Carbohydrates; Cell physiology; Cells; Chemical Dynamics; Chemicals; Chemistry; Collaborations; Complex; Custom; Denmark; Development; Disease; Genome; Glycopeptides; Glycoside Hydrolases; Grant; Human; Immune response; Isotopically-Coded Affinity Tagging; Kinetics; Lead; Letters; Libraries; Light; Link; Malignant Neoplasms; Manuals; Mass Spectrum Analysis; Measures; Methods; Microarray Analysis; Modeling; Modification; Molecular; Pattern; Peptide Library; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phase; Phosphorylation; Phosphotransferases; Play; Polysaccharides; Post-Translational Protein Processing; Process; Protein Glycosylation; Protein Microchips; Proteins; Proteome; Proteomics; Provider; Reagent; Receptor Cell; Regulation; Reporting; Reproducibility; Resolution; Role; Screening procedure; Serum; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Techniques; Technology; Tissues; Universities; Virus; Work; base; carbohydrate structure; cell motility; combinatorial; commercialization; density; disease diagnosis; glycosylation; improved; instrumentation; interest; member; novel; novel diagnostics; novel strategies; professor; prognostic; protein degradation; protein folding; protein function; protein structure; prototype; research study

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). PUBLIC HEALTH RELEVANCE: Proteins, the functional machinery of cells, are tightly regulated by the cell using hundreds of possible dynamic, chemical modifications to the protein's structure, including by the attachment of carbohydrate molecules (glycosylation). In disease processes such as cancer, these regulatory mechanisms become dysfunctional. Here we propose to develop improved technology for measuring these changes which combines light cleavable chemical linkers, advanced mass spectrometry techniques and protein (peptide) microarrays which will ultimately lead to a better understanding of disease mechanisms and how to detect and treat diseases.

描述：蛋白质翻译后修饰 (PTM) 在多种细胞过程中发挥着核心作用，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。已知超过 300 种不同类型的 PTM，估计存在于所有蛋白质的 80% 中，部分原因是与基因组相比，蛋白质组要大得多。人们越来越认识到表征这些 PTM 以及它们如何调节蛋白质功能的重要性对于理解疾病的分子基础、发现新的诊断/预后生物标志物、开发新的药物疗法，甚至理解相互作用至关重要。具有细胞受体的不同病毒。然而，开发有效的技术来检测和分析可能非常复杂的 PTM，尤其是在蛋白质糖基化的情况下，存在许多挑战。正如本次资助申请中所述，“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）筛选糖组 PTM 方面取得了进展。然而，蛋白质微阵列技术的局限性，包括相对较低的密度（尤其是在排列大型蛋白质/肽文库时）、较差的重现性和较差的测定动力学，使得这种方法不太理想。此外，与传统上用于分析肽和蛋白质糖基化的质谱法不同，微阵列不提供此类信息。糖肽的大型组合珠文库提供了微阵列的替代方案，但通常利用“淘选”方法来测量与文库的相互作用，需要手动“挑选”大的单个珠子，以便随后通过质谱进行逐一分析。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 开发的光裂解连接技术的优点。在一个示例中，将合成可光裂解的糖肽珠库并将其随机掺入高密度皮孔板中以形成阵列。正如初步实验所证明的，这种方法可以快速测量珠阵列中潜在数百万种不同“诱饵”糖肽上相互作用的生物分子（例如聚糖结合蛋白（GBP）、糖苷酶/糖转移酶、激酶和药物）的影响，高灵敏度和空间分辨率。在第二个非基于阵列的示例中，用含有特定“猎物”类型的感兴趣的生物样本（例如血清自身抗体）处理可光裂解的糖肽珠库。然后使用“光释放和重新捕获”工作流程将糖肽-猎物复合物快速光富集至非常高的纯度。随后进行传统的基于质谱的蛋白质组学分析，以识别相互作用的诱饵糖肽，从而快速识别用于疾病诊断和治疗的潜在生物标志物。第三种方法基于最近报道的使用 AmberGen 的光裂解接头来识别细胞、组织和其他生物样本中的 O-连接 β-N-乙酰氨基葡萄糖 (O-GlcNAc) 蛋白质修饰。这些修饰的重要性已与磷酸化相比较，但我们准确检测和表征它们的能力刚刚通过令人兴奋的新方法出现。在这里，我们将改进这些方法，使用专有的光裂解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。为了加速该项目所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（马萨诸塞州比尔里卡）密切合作（参见 Gary Kruppa 博士的来信） ，业务开发副总裁）。此外，我们还将与丹麦哥本哈根大学糖组学中心的 Ola Blixt 博士（糖肽文库合成稳健方法的开发者）和波士顿大学生物医学质谱中心主任、主席 Cathy Costello 博士合作，人类蛋白质组组织，生物化学、生物物理学和化学教授，是基于质谱的糖组学技术领域公认的专家（参见 Blixt 博士和科斯特洛）。 公共健康相关性：蛋白质是细胞的功能机制，受到细胞对蛋白质结构进行数百种可能的动态化学修饰的严格调节，包括通过碳水化合物分子的附着（糖基化）。在癌症等疾病过程中，这些调节机制会变得功能失调。在这里，我们建议开发改进的技术来测量这些变化，该技术结合了光可裂解化学接头、先进的质谱技术和蛋白质（肽）微阵列，最终将有助于更好地了解疾病机制以及如何检测和治疗疾病。