Unified Process for the Discovery of Protein Biomarkers and their X-Aptamer Affinity Reagents

发现蛋白质生物标志物及其 X 适体亲和试剂的统一流程

• 批准号: 10091672
• 负责人: Lokesh Rao
• 金额: $ 11.87万
• 依托单位: AM BIOTECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091672
• 关键词: Unified; Process; Discovery; Protein; Biomarkers

ABSTRACT Reflecting a particular physiological state, biomarkers are very important for earlier diagnosis of diseases, realizing personalized medicine, as well as facilitating drug development and discovery. For these reasons, significant research efforts are currently being directed towards biomarker discovery and transitioning them into clinical use. AM Biotech is developing a new discovery proteomics platform called “Unified Process for Discovering Protein Biomarkers and their X-Aptamers” (Unified Process). This technology is a unique proteomics tool created from the combination of AM Biotech's bead-based X-Aptamer selection platform, multi- color flow cytometry, and liquid chromatography tandem mass spectrometry. This Unified Process is highly attractive in that it allows for multiplexing, detection of low abundance differences and relatively quick and efficient processing. However, most importantly, it integrates the major stages of the protein biomarker pipeline: 1) discovery (sometimes separated into candidate identification and prioritization), 2) verification, and 3) clinical validation into a more efficient pipeline. Unlike typical protein biomarker discovery techniques such as “shotgun” proteomics and 1D/2D gels coupled with MS, which includes difference gel electrophoresis (DIGE), the Unified Process identifies an X-Aptamer (XA) affinity reagent for every candidate biomarker. The availability of an affinity reagent to each candidate biomarker from the earliest stage of discovery is transformative to the pipeline because there are no assays for the vast majority of human proteins and such assays must be developed de novo. The XAs, selected against native proteins in their native environment enable multiplexed downstream assays, protein concentration from samples for more precise identification of the candidates using MS, and the ability to quickly develop an affinity-enrichment multiple reaction monitoring MS assay (AEMRM MS). The Unified Process consists of four key steps, and of these, the most important of which is the initial flow cytometry analysis of beads bound to differentially labeled proteomes. To date, AM Biotech has established flow cytometry conditions that will allow differences between two proteomes labeled with different fluorophores to be analyzed. In this application, an optimized 4-color process will be developed. This will require several inter-related variables to be studied. Once conditions for 4-color sorting are established, spiking experiments will be used to characterize the performance of the process. Successful completion of the specific aims will pave the way for a Phase II investigation to integrate, optimize and fully characterize the remaining steps of the Unified Process and to use the process to analyze patient plasma samples.

摘要 生物标志物反映了特定的生理状态，对于早期诊断非常重要。 疾病，实现个性化医疗，以及促进药物开发和发现。为这些 由于这些原因，重要的研究工作目前正致力于生物标志物的发现和转化， 将其用于临床。AM Biotech正在开发一种新的发现蛋白质组学平台，称为“统一过程 用于发现蛋白质生物标记物及其X-适体”（统一过程）。这项技术是独一无二的 该蛋白质组学工具结合了AM Biotech基于珠的X-Aptamer选择平台、多 彩色流式细胞术和液相色谱串联质谱法。 这种统一的过程是非常有吸引力的，因为它允许多路复用，检测低丰度 差异和相对快速和有效的处理。然而，最重要的是， 蛋白质生物标志物管道的阶段：1）发现（有时分为候选物鉴定 和优先级）、2）验证和3）临床确认到更有效的管道中。不同于典型的蛋白质 生物标志物发现技术，如“鸟枪”蛋白质组学和与MS结合的1D/2D凝胶， 包括差异凝胶电泳（DIGE），统一过程鉴定X-适体（XA）亲和力 每个候选生物标志物的试剂。亲和试剂对每种候选生物标志物的可用性 从发现的最早阶段到管道都是变革性的，因为没有针对大量物质的分析 大多数人蛋白质和这样的测定必须从头开发。相对于本机 蛋白质在其天然环境中能够进行多重下游测定， 使用MS更精确地识别候选人的样品，以及快速开发 亲和富集多反应监测MS测定（AEMRMS）。 统一流程由四个关键步骤组成，其中最重要的是初始 与差异标记的蛋白质组结合的珠子的流式细胞术分析。到目前为止，AM Biotech已 建立了流式细胞术条件，这将允许用不同标记的两种蛋白质组之间的差异 待分析的荧光团。在此应用中，将开发优化的四色工艺。这将 需要研究几个相互关联的变量。一旦建立了四色分选的条件， 将使用实验来表征该工艺的性能。成功完成具体的 目标将为第二阶段的研究铺平道路，以整合，优化和充分表征剩余的 统一过程的步骤，并使用该过程来分析患者血浆样品。