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

描述(申请人提供)：人类基因组的测序已经导致了一个新的、甚至更雄心勃勃的目标--人类蛋白质组的特征。这样的努力不仅涉及了解在人类细胞中表达的数十万种不同蛋白质的功能，还涉及描述可能与其他细胞和细胞外分子发生的数百万种潜在相互作用，包括蛋白质、核酸、脂类和小分子。快速进行如此大规模的全球蛋白质组筛选的能力将成为许多癌症研究领域的强大工具，如生物标记物发现、绘制细胞网络图和药物开发。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，但高密度蛋白质微阵列尚未产生类似的影响。目前蛋白质芯片技术的局限性包括阵列密度低、重复性差、成本高、检测动力学差、难以检测各种诱饵-猎物相互作用以及酶诱导的蛋白质修饰。相比之下，传统蛋白质组学中使用的质谱学确实提供了许多这样的能力，包括对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，与传统的基于质谱学的蛋白质组学结合使用的分离方法，如二维凝胶电泳和液相色谱，速度很慢，而且远不如微阵列中固有的蛋白质物理排列/分类那样健壮。在第一阶段，我们将评估AmberGen开发的一种新的蛋白质组学方法，称为基于珠的全球蛋白质组筛选(Bead-GPSTM)，它结合了MALDI质谱学成像(MALDI-MSI)和微阵列技术的优点。这种方法利用可光裂解的质量标签(PC-质量标签)来编码蛋白质珠库(诱饵库)以及相互作用的猎物分子，如其他蛋白质，所有这些都显示在以高密度(1,000,000个孔)随机排列的单个珠子上。由于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，因此有可能对诱饵-猎物相互作用进行高度多重筛选，远远超出传统荧光微阵列的能力。然而，荧光成像仍然可以与Bead-GPS一起使用，以预先识别和量化积极的相互作用，然后由MALDI-MSI解码。此外，由于MALDI-MSI能够对i)相互作用的猎物分子(如小药物化合物)、ii)其他蛋白质(蛋白质片段指纹图谱)和iii)蛋白质修饰(例如丝氨酸或酪氨酸磷酸化)进行珠上无标记检测，从而进一步扩展了Bead-GPSTM的能力。在第一阶段，我们将使用无细胞蛋白质翻译技术构建一个100个成员的原型蛋白质珠库，以评估Bead-GPSTM的关键功能，包括PC-质量标签编码(对于诱饵和猎物分子)，使用PC-质量标签和通过无标签方法进行蛋白质-蛋白质相互作用分析，检测无标签蛋白质-药物相互作用，检测蛋白质修饰和血清图谱，以发现癌症生物标记物。在第二阶段，将建造和测试一个完整的蛋白质组范围的Bead-GPS“平台。为了加速该项目产生的产品的商业化，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，但在蛋白质组学领域尚未出现类似的影响，尽管有高密度商业蛋白质微阵列的存在。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筛选(Bead-GPSTM)，它结合了MALDI质谱学成像和微阵列的优势，克服了蛋白质组技术中现有的局限性。新方法的潜在好处包括发现癌症诊断的新生物标记物，增加对癌症原因的了解，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.