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

基本信息

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
项目类别：
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

项目摘要

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：胰腺癌目前是美国第三大癌症死亡原因，有 5- 年生存率<5%。大多数可能治愈的患者都患有不可切除的局部晚期标准治疗包括化疗和放射治疗相结合的疾病。一个专业胰腺癌治疗中的挑战是治疗反应的早期评估。小说迫切需要早期治疗反应的标志物，以便就类型做出更明智的决策考虑到与这些治疗相关的高毒性，以及治疗的顺序。在我们之前的工作中，我们能够从局部晚期胰腺癌患者的血清中鉴定出 20 多种外泌体蛋白标记物通过使用外泌体分离方法和质谱分析来治疗期间的癌症。然而，这种针对大型临床队列的分析受到外泌体制备的重复性的限制。外泌体蛋白的准确定量是外泌体组学研究领域的一个重要问题。在当前的提案中，我们计划重点开发外泌体分离和表征的分析关于严谨性和可重复性。我们将开发一种基于超级 SILAC（氨基稳定同位素标记）细胞培养中的酸）外泌体方法作为通用内标（UIS）。原则上，当超级SILAC- 将基于外泌体的外泌体掺入人血清中进行蛋白质组分析，它可以遵循相同的实验工作流程作为血清外泌体，可以最大限度地减少样品制备和 LC-MS 引入的变化（液相色谱-质谱）分析。因此，SILAC 标记的外泌体将是具有双重功能的理想选择。用于生物标志物分析的功能，用于可重复的样品制备并充当 UIS。靶向 LC-SRM 和我们之前研究中的标记物 PRISM-SRM 外泌体分析将与 UIS 一起使用以精确监测化学放射治疗过程中蛋白质的变化。局部晚期胰腺癌患者癌症将在化疗之前、期间和之后进行连续抽血，然后进行化学放射。然后我们将进行基于外泌体标记的靶向蛋白质组学验证研究我们在之前的工作中发现，对化疗放疗过程有特定反应。标记与基线相比的值变化将与治疗结果相关（例如无进展患者的生存率和总生存率）。这项工作将确定潜在的用途和测量这些外泌体标记物的可重复性，用于监测治疗期间和治疗后蛋白质标记物的变化。本文使用的基于 LC-SRM 质谱的测定具有明显的优点，即可以多路复用超过同时 20 个标记。所提出的工作包含几个新颖的方面，包括：1）使用超级 SILAC 解决外泌体蛋白精确定量的不重复性问题，特别是多步骤外泌体制备和 2) 多重靶向 LC-SRM 和高灵敏度 PRISM-SRM 检测患者血清外泌体的生物标志物分析。