A Proteomics Platform for Quantitative, Ultra-High Throughput, and Ultra-Sensitiv

描述(由申请人提供)： 该项目的总体目标是开发一个癌症生物标记物发现和验证平台，用于对人体血浆和其他临床相关样本的分析，将提供以下测量：更可靠、更高灵敏度、提供超过数量级的吞吐量，并与现有平台相比具有更高的定量效用。新平台将提供更大的动态范围，从而扩大蛋白质组覆盖范围，并允许在目前对广泛蛋白质组测量有问题的浓度(例如，与PSA相似或更低的血浆蛋白质浓度)对候选癌症生物标记蛋白进行定量测量。平台吞吐量的提高将与定量数据质量的提高(来自更高的稳健性、更高的跑到跑的重复性和更高的灵敏度)一起实现，从而对蛋白质组数据提供更大的信心。因此，新平台将成为候选生物标记物发现和临床前候选验证的有力工具，并随着进一步的完善为广泛的临床应用提供基础。该仪器平台将是基于快速液相色谱(LC)的分离、新型微制造阵列纳米电喷雾电离(NanESI)界面、高分辨率气相离子迁移率分离与准确的飞行时间质谱学的集成。在R21的初始阶段，我们将主要关注：(1)开发原型仪器平台和关键的NanESI接口组件；以及(2)对平台的吞吐量、灵敏度和健壮性进行初步评估。R33阶段将集中于：(1)平台的进一步改进，(2)支持平台海量数据生产速度所需的信息学工具的开发，以及(3)平台在高通量定量蛋白质组学测量的背景下的评估，该测量涉及临床相关的基于肿瘤学的人体血浆样本，并与一些低水平蛋白质生物标志物(例如PSA)的现有分析进行比较。该项目的预期结果将是一个强大的高通量平台，具有革命性的癌症生物标记物发现和验证及其临床应用的初步传播的潜力。

DESCRIPTION (provided by applicant): The overall objective of this project is to develop a cancer biomarker discovery and validation platform for analyses e.g. of human blood plasma and other clinically relevant samples that will provide measurements that are: much more robust, of higher sensitivity, provide more than order of magnitude throughput, and have improved quantitative utility compared to existing platforms. The new platform will provide increased dynamic range, and thus proteome coverage, and allow quantitative measurements of candidate cancer biomarker proteins at concentrations that are presently problematic for broad proteome measurements (e.g. plasma protein concentrations similar to PSA and lower). The enhanced throughput of the platform will be realized in conjunction with improved quality of quantitative data (derived from increased robustness, higher run-to-run reproducibility, and increased sensitivity), providing greater confidence in proteomic data. Thus, the new platform will be a powerful tool for both candidate biomarker discovery and pre-clinical candidate validation, and with further refinement provide the basis for broad clinical application. The instrumental platform will be an integration of fast liquid chromatography (LC) based separations, a novel microfabricated array nanoelectrospray ionization (nanoESI) interface, and high resolution gas phase ion mobility separations coupled with accurate mass time-of-flight mass spectrometry. In the initial R21 phase, we will focus primarily on: (1) the development of a prototype instrument platform and the key nanoESI interface component; and (2) an initial evaluation of the platform's throughput, sensitivity, and robustness. The R33 Phase will focus on: (1) the further refinement of the platform, (2) the development of the informatics tools needed to support the platform's massive data production rates, and (3) the platform's evaluation in the context of high throughput quantitative proteomics measurements involving clinically relevant oncology-based human blood plasma samples and the comparison with existing assays for a number of low level protein biomarkers (e.g. PSA). The anticipated result of this project will be a robust high throughput platform with the potential for revolutionizing cancer biomarker discovery and validation, and its initial dissemination for clinical applications.