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

描述（由申请人提供）：人类基因组测序已经导致了一个新的，甚至更雄心勃勃的目标-人类蛋白质组的表征。这样的奋进不仅涉及了解人类细胞中表达的数十万种不同蛋白质的功能，还涉及表征可能与其他细胞和细胞外分子（包括蛋白质、核酸、脂质和小分子）发生的数百万种潜在相互作用。快速进行如此大规模的全球蛋白质组筛选的能力将成为癌症研究许多领域的强大工具，如生物标志物发现，细胞网络映射和药物开发。虽然近20年前推出的高密度DNA微阵列对促进基因组革命产生了重大影响，但高密度蛋白质微阵列尚未产生类似的影响。目前蛋白质微阵列技术的局限性包括阵列密度低、再现性差、成本高、测定动力学差和难以检测多种诱饵-猎物相互作用以及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确实提供了许多这些能力，包括小药物化合物的无标记鉴定、蛋白质修饰的鉴定和蛋白质鉴定。然而，与常规的基于质谱的蛋白质组学（如二维凝胶电泳和液相色谱）结合使用的分离方法是缓慢的，并且不如微阵列中固有的蛋白质的物理排列/分选那样稳健。在第一阶段，我们将评估AmberGen为蛋白质组学开发的一种新方法，称为基于珠的全球蛋白质组学筛选（Bead-GPSTM），该方法结合了MALDI质谱成像（MALDI-MSI）和微阵列技术的优势。该方法利用光可裂解的质量标签（PC-Mass-Tags）来编码蛋白质-珠文库（诱饵文库）以及相互作用的猎物分子如其他蛋白质，所有这些都展示在Pico-孔板中随机排列在高密度（1，000，000威尔斯）处的单个珠上。因为我们已经在初步实验中表明，高密度蛋白质微珠阵列的MALDI-MSI具有快速识别数百万种不同质量标签组合的潜力，具有高灵敏度和空间分辨率，因此可以对诱饵-猎物相互作用进行远远超出常规荧光微阵列能力的高度多重筛选。然而，荧光成像仍然可以与Bead-GPS一起使用，以预先鉴定和定量阳性相互作用，然后通过MALDI-MSI解码。此外，通过MALDI-MSI对以下各项进行珠上无标记检测的能力进一步扩展了Bead-GPSTM的能力：i）相互作用的猎物分子，例如小药物化合物，ii）其他蛋白质（蛋白质片段化指纹）和iii）蛋白质修饰（例如丝氨酸或酪氨酸磷酸化）。在第一阶段，我们将使用无细胞蛋白质翻译技术构建100个成员的原型蛋白质-珠库，以评估Bead-GPSTM的关键特征，包括PC-质量标签编码（对于诱饵和猎物分子），用PC-Mass-Tags和通过无标记手段的蛋白质-蛋白质相互作用分析，无标记蛋白质-药物相互作用的检测，用于发现癌症生物标志物的蛋白质修饰检测和血清分析。在第二阶段，将构建和测试一个完整的蛋白质组范围的Bead-GPS平台。为了加速该项目产品的商业化，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，开发一种用户友好的完全集成的仪器（和软件），作为Bead-GPS技术的平台。 公共卫生相关性：尽管近20年前引入的高密度DNA微阵列在促进基因组革命方面产生了重大影响，但尽管高密度商业蛋白质微阵列的可用性，在蛋白质组学领域还没有发生类似的影响。我们将在第一阶段评估一种新的蛋白质组学方法，称为基于珠的全球蛋白质组学筛选（珠-GPSTM），通过结合MALDI质谱成像和微阵列的优势，克服了蛋白质组学技术的现有局限性。新方法的潜在好处包括发现新的癌症诊断生物标志物，增加对癌症病因的了解以及发现治疗癌症的新药。

DESCRIPTION (provided by applicant): Sequencing of the human genome has led to a new and even more ambitious goal - characterization of the human proteome. Such an endeavor involves not only understanding the function of hundreds of thousands of different proteins expressed in human cells but also characterizing the millions of potential interactions that can occur with other cellular and extracellular molecules including proteins, nucleic acids, lipids and small molecules. The ability to rapidly perform such massive global proteomic screens would be a powerful tool in many areas of cancer research such as biomarker discovery, mapping cellular networks and drug development. Although high-density DNA microarrays introduced almost 20 years ago have had a major impact in facilitating the genomic revolution, high-density protein microarrays have not yet exerted a similar impact. Current limitations in protein microarray technology include low array density, poor reproducibility, high cost, poor assay kinetics and difficulty in detecting a diversity of bait-prey interactions as well as enzyme-induced protein modifications. In contrast, mass spectrometry used in conventional proteomics does provide many of these capabilities including label-free identification of small drug compounds, identification of protein modifications and protein identification. However, the separation methods used in conjunction with conventional mass spectrometry based proteomics such as two-dimensional gel electrophoresis and liquid chromatography are slow and not nearly as robust as the physical arraying/sorting of proteins inherent in a microarray. During Phase I we will evaluate a new approach developed by AmberGen for proteomics termed Bead-based Global Proteomic Screening (Bead-GPSTM) which combines the advantages of MALDI mass spectrometry imaging (MALDI-MSI) and microarray technology. This approach utilizes photocleavable mass-tags (PC-Mass-Tags) to encode a protein-bead library (bait library) as well as interacting prey molecules such as other proteins, all displayed on individual beads randomly arrayed at high-density (1,000,000 wells) in a Pico-well plate. Because we have shown in preliminary experiments that MALDI-MSI of high density protein-bead arrays has the potential to rapidly identify millions of different mass-tag combinations, with high sensitivity and spatial resolution, it is possible to perform highly multiplexed screening of bait-prey interactions far beyond the capabilities of conventional fluorescence microarrays. However, fluorescence imaging can still be used with Bead-GPS" to pre- identify and quantitate positive interactions which are then decoded by MALDI-MSI. In addition, the power of Bead-GPSTM is further extended by the ability of MALDI-MSI to perform on-bead label-free detection of i) interacting prey molecules such as small drug compounds, ii) other proteins (protein fragmentation fingerprinting) and iii) protein modifications (e.g. serine or tyrosine phosphorylation). During Phase I we will fabricate a 100-member prototype protein-bead library using cell-free protein translation techniques in order to evaluate key features of Bead-GPSTM including PC-Mass-Tag coding (for both bait and prey molecules), protein-protein interaction analysis both with PC-Mass-Tags and by label-free means, detection of label-free protein-drug interactions, detection of protein modifications and serum profiling for cancer biomarker discovery. During Phase II, a full proteome-wide Bead-GPS" platform will be constructed and tested. In order to accelerate commercialization of the 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, to develop a user- friendly, fully integrated instrument (and software) which will serve as a platform for the Bead-GPS" technology. PUBLIC HEALTH RELEVANCE: Although high density DNA microarrays introduced almost 20 years ago have had a major impact in facilitating the genomic revolution, a similar impact has not yet occurred in the field of proteomics despite the availability of high density commercial protein microarrays. We will evaluate in Phase I a new approach for proteomics termed Bead- based Global Proteomic Screening (Bead-GPSTM) which overcomes existing limitations in proteomic technology by combining the advantages of MALDI mass spectrometric imaging and microarrays. Potential benefits of the new approach include the discovery of new biomarkers for cancer diagnostics, increased understanding of the causes of cancer and discovery of new drugs to treat cancer.