Universal Internal Standard for Reproducible Accurate Quantification of Exosome Protein Markers

用于外泌体蛋白标记物可重复准确定量的通用内标

• 批准号: 10358672
• 负责人: David M. Lubman
• 金额: $ 46.69万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-14 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10358672
• 关键词: Address; Aftercare; Amino Acids; Biological Assay; Biological Markers; Blood; Caliber; Cancer Etiology; Cancer cell line; Cell Culture Techniques; Cell Line; Cells; Cessation of life; Chemotherapy and/or radiation; Clinical; Combination Drug Therapy; Detection; Disease; Early treatment; Future; Human; Knowledge; Label; Liquid Chromatography; Malignant neoplasm of pancreas; Mass Chromatography; Mass Spectrum Analysis; Measurement; Membrane; Methods; Monitor; Nature; Normal Cell; Patient-Focused Outcomes; Patients; Performance; Population; Preparation; Prognosis; Progression-Free Survivals; Prospective cohort; Protein Analysis; Proteins; Proteomics; Radiation therapy; Reproducibility; Research; Sampling; Serum; Serum Markers; Stable Isotope Labeling; Survival Rate; Therapeutic; Toxic effect; Treatment outcome; Unresectable; Variant; Vesicle; Work; X-Ray Computed Tomography; advanced disease; advanced pancreatic cancer; assay development; base; biomarker panel; biomarker validation; cancer therapy; cancer type; chemoradiation; chemotherapy; cohort; exosome; imaging modality; improved; individual patient; microvesicles; minimally invasive; multiplex assay; nanosized; neoplastic cell; novel; novel marker; pancreatic cancer cells; pancreatic cancer patients; protein biomarkers; response; standard of care; tool; treatment response; tumor

Abstract: Pancreatic cancer is currently the third leading cause of cancer related to death in the USA with a 5- year survival rate of <5%. Most potentially curable patients present with unresectable locally advanced disease where the standard of care includes a combination of chemotherapy and radiation therapy. A major challenge in the management of pancreatic cancer is the early assessment of treatment response. Novel markers of early treatment response are critically needed to make better informed decisions regarding the type and sequencing of therapies, given the high toxicity associated with these treatments. In our previous work we were able to identify 20+ exosomal protein markers from the serum of patients with locally advanced pancreatic cancer during therapeutic treatment by using exosome isolation methods and mass spectrometry analysis. However, such analysis for large clinical cohorts was limited by the reproducibility in exosome preparation for accurate quantification of exosomal proteins, which is a significant problem in the exosome omics research field. In the current proposal, we plan to develop the analytics of exosome isolation and characterization with a focus on rigor and reproducibility. We will develop a super-SILAC-based (Stable Isotope Labeling by/with Amino acids in Cell culture) exosome method as a universal internal standard (UIS). In principle, when the super-SILAC- based exosome is spiked into human serum for proteomic analysis it can follow the same experimental workflow as serum exosomes which could maximally reduce variations introduced by sample preparation and LC-MS (Liquid Chromatography-Mass Spec) analysis. Therefore, SILAC-labeled exosomes will be ideal with a dual function for biomarker analysis for reproducible sample preparation and acting as a UIS. Targeted LC-SRM and PRISM-SRM exosome analyses of markers from our prior studies will be used with the UIS to precisely monitor changes in proteins during a course of chemo-radiation therapy. Patients with locally advanced pancreatic cancer will undergo serial blood draws prior to, during, and after treatment with chemotherapy followed by chemo-radiation. We will then perform a targeted proteomics confirmation study based on exosome markers identified in our previous work that have a specific response to a course of chemo-radiation therapy. The marker value changes compared to baseline will be associated with the treatment outcomes (such as progression-free survival and overall survival) in patients. This work will establish the potential use and measurement reproducibility of these exosomal markers for monitoring changes in protein markers during and after treatment. The LC-SRM mass spec-based assay to be used herein has the distinct advantage of being multiplexed for over 20 markers simultaneously. The proposed work contains several novel aspects including: 1) the use of super- SILAC to address irreproducibility in accurate quantification of exosome proteins especially for multi-step exosome preparation and 2) multiplexed targeted LC-SRM and highly sensitive PRISM-SRM assays for biomarker analysis in patient serum exosomes.

摘要：胰腺癌目前是美国与死亡有关的癌症的第三主要原因，5-- 年生存率<5％。大多数可治愈的患者存在于本地不可切除的患者 护理标准包括化学疗法和放射治疗的结合。专业 胰腺癌管理的挑战是治疗反应的早期评估。小说 早期治疗反应的标志需要至关重要 鉴于与这些治疗相关的高毒性，疗法的测序。在我们以前的工作中 能够从局部晚期胰腺患者的血清中鉴定20多个外泌体蛋白质标记 通过使用外泌体分离方法和质谱分析，治疗过程中的癌症。 但是，大型临床队列的这种分析受外部准备的可重复性限制 准确的外泌体蛋白质定量，这在外泌体法学研究领域是一个重要的问题。 在当前的建议中，我们计划以重点进行外部隔离和表征的分析 严格和可重复性。我们将开发一个基于超锡的（稳定的同位素标签） 细胞培养中的酸）外泌体法作为通用的内标（UI）。原则上，当超级硅胶 - 基于基于蛋白质组学分析的人血清中的外泌体可以遵循相同的实验工作流程 作为血清外泌体，可以最大程度地减少样品制备和LC-MS引入的变化 （液相色谱 - 质量规格）分析。因此，Silac标记的外泌体将是理想的选择 生物标志物分析的功能，用于可再现样品制备和充当UI。靶向LC-SRM和 Prism-SRM对我们先前研究的标记的外部分析将与UI一起使用，以精确监测 在化学放射治疗过程中蛋白质的变化。局部晚期胰腺的患者 癌症将在化学疗法之前，期间和治疗后接受系列血液。 化学辐射。然后，我们将基于外泌体标记进行靶向蛋白质组学确认研究 在我们以前的工作中确定，对化学放射治疗方案有特定的反应。标记 与基线相比的价值变化将与治疗结果有关（例如无进展 患者的生存和整体生存）。这项工作将确定潜在的使用和测量 这些外泌体标记物可重现用于监测治疗过程中蛋白质标记的变化的可重复性。 本文使用的基于LC-SRM质量规格的测定具有明显的优势 同时20个标记。提出的工作包含几个新方面，包括：1）使用超级 SILAC以准确量化外泌体蛋白质，特别是用于多步的准确量化。 外泌体制备和2）多路复用的靶向LC-SRM和高度敏感的Prism-SRM分析 患者血清外泌体中的生物标志物分析。