Global Proteomic Screening by MALDI Spectrometric Imaging of Protein-Bead Arrays

描述（由申请人提供）：近25年前高密度DNA微阵列的出现是基因组学革命的重要一步。主要的成功包括全基因组基因表达谱（GEP），这导致了对细胞控制途径的新理解和强大的诊断/预后测试，如预测乳腺癌复发。原则上，类似的方法，即利用高密度蛋白质微阵列，可能预示着蛋白质组学的革命，特别是在癌症研究领域。 潜在的应用包括测量蛋白质-蛋白质相互作用、旨在发现肿瘤自身抗体生物标志物的免疫应答分析以及分析参与信号转导的酶底物相互作用（例如激酶-底物相互作用）。然而，尽管不断取得进展，高密度蛋白质微阵列尚未实现其最初的承诺。目前的局限性包括低阵列密度，再现性差，成本高，不良的测定动力学和难以检测的多样性诱饵-猎物的相互作用，包括酶诱导的蛋白质修饰。相比之下，在常规蛋白质组学中使用的质谱法提供了许多重要的能力，包括通过测序（和指纹）进行蛋白质鉴定以及蛋白质修饰（例如磷酸化）的分子水平鉴定，但不具有微阵列的并行化和高通量能力。在第一阶段，我们评估了AmberGen开发的一种新蛋白质组学方法的可行性，该方法称为基于珠子的全球蛋白质组学筛选（Bead-GPS）。该方法结合了质谱（MS）和微阵列技术的优点，利用相关MALDI质谱成像（MALDI-MSI）和荧光成像的光可裂解的质量标签编码的随机微珠阵列。实现了所有具体目标，包括：i）展示珠-GPS同步荧光和MALDI-MS成像; ii）展示蛋白质/肽珠文库的高容量编码; iii）开发改进的光可切割接头; iv）开发用于珠阵列的改进的微孔基底; v）证明使用Bead-GPS筛选由光可裂解肽/仿生肽组成的大型组合合成的一珠/一化合物（OBOC）珠文库的可行性。在第二阶段，我们将进一步优化Bead-GPS”的主要组成部分，包括构建肽和类肽的光可裂解珠库，将库格式化为高密度随机珠阵列，并获得珠阵列的相关荧光和MALDI-MS图像，以识别和解码“猎物”分子与珠库（“诱饵”）的生物分子相互作用。具体的重点将是验证珠GPS”的两个应用程序有关的癌症研究和临床诊断。第一个应用涉及使用Bead-GPS筛选中等大小的平行合成的光可裂解肽珠文库，然后筛选用于激酶底物相互作用的更大的组合合成的OBOC文库。第二个应用是使用Bead-GPS”来针对患者血清筛选大的组合OBOC仿生肽文库（类肽），以发现可用作癌症免疫测定中的生物标志物的替代抗原。为了加速珠GPS的开发和商业化”，AmberGen将与罗恩祖克曼博士密切合作，他是伯克利分子铸造厂生物纳米结构设施的主任，也是仿生肽合成领域公认的先驱; Cathy Costello博士，主任，波士顿大学医学院生物医学质谱中心，MALDI及其生物医学应用领域的领先专家;加里克鲁帕博士，布鲁克道尔顿公司（马萨诸塞州比勒里卡）业务发展副总裁，世界领先的MALDI-MS仪器供应商;保罗比林斯博士，生命技术公司的首席医疗官，生物技术产品和服务的营销领先公司。

DESCRIPTION (provided by applicant): The advent of high density DNA microarrays almost 25 years ago has been an essential step in the genomics revolution. Major successes include genome-wide gene expression profiling (GEP) which has led to a new understanding of cellular control pathways and powerful diagnostic/prognostic tests such as for predicting breast cancer recurrence. In principle, a similar approach, namely the utilization of high density protein microarrays, could herald a revolution in proteomics, particularly in the field of cancer research. Potential applications include measuring protein-protein interactions, immune response profiling aimed at discovery of tumor autoantibody biomarkers and profiling enzyme substrate interactions involved in signal transduction (e.g. kinase-substrate interactions). However, despite continued progress, high-density protein microarrays have not yet lived up to their original promise. Current limitations include low array density, poor reproducibility, high cost, poor assay kinetics and difficulty in detecting a diversity of bait-prey interactions including enzyme-induced protein modifications. In contrast, mass spectrometry used in conventional proteomics provides many important capabilities including protein identification by sequencing (and fingerprinting) as well as molecular-level identification of protein modifications (e.g. phosphorylation), yet does not have the parallelization and high throughput capacity of microarrays. During Phase I we evaluated the feasibility of a new proteomic approach developed by AmberGen termed Bead- based Global Proteomic Screening (Bead-GPS"). This method combines the advantages of mass spectrometry (MS) and microarray technology by using correlated MALDI mass spectrometric imaging (MALDI-MSI) and fluorescence imaging of photocleavable Mass-Tag-coded random bead-arrays. All of the specific aims were achieved including: i) Demonstration of Bead-GPS" synchronized fluorescent and MALDI-MS imaging; ii) Demonstration of high capacity coding of protein/peptide bead libraries; iii) Development of improved photocleavable linkers; iv) Development of improved micro-well substrates for the bead-arrays; v) Demonstration of the feasibility to use Bead-GPS" to screen large combinatorial synthesized one-bead/one-compound (OBOC) bead-libraries comprised of photocleavable peptides/biomimetic peptides. During Phase II, we will further optimize major components of Bead-GPS" including the construction of photocleavable bead-libraries of peptides and peptoids, formatting the libraries into high density random bead- arrays and obtaining correlated fluorescence and MALDI-MS images of the bead-arrays to identify and decode bio- molecular interactions of "prey" molecules with the bead-library (the "bait"). Specific emphasis will be on validating Bead-GPS" for two applications relevant for cancer research and clinical diagnosis. The first application involves use of Bead-GPS" to screen mid-size, parallel synthesized, photocleavable peptide bead-libraries and then larger combinatorial synthesized OBOC libraries for kinase substrate interactions. The second application is the use of Bead-GPS" to screen large combinatorial OBOC biomimetic peptide libraries (peptoids) against patient sera for the discovery of surrogate antigens that can be used as biomarkers in cancer immunoassays. In order to accelerate the development and commercialization of Bead-GPS", AmberGen will work a closely with Dr. Ron Zuckermann, Director of the Biological Nanostructure Facility at the Molecular Foundry located at Berkeley and a recognized pioneer in the field of biomimetic peptide synthesis; Dr. Cathy Costello, Director, Center for Biomedical Mass Spectrometry at Boston University Medical School and a leading expert in the field of MALDI and its biomedical applications; Dr. Gary Kruppa, V.P. of Business Development at Bruker Daltonics (Billerica, MA), a world-leading provider of MALDI-MS instrumentation; and Dr. Paul Billings, the Chief-Medical Officer at Life Technologies, a leading company in marketing biotechnology products and services.