Universal Internal Standard for Reproducible Accurate Quantification of Exosome Protein Markers

翻译后摘要：胰腺癌是目前的第三大癌症相关的死亡原因，在美国与5- 年生存率<5%。大多数潜在可治愈的患者存在不可切除的局部晚期 标准治疗包括化疗和放疗联合治疗的疾病。一个主要 胰腺癌管理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定。 和治疗顺序，考虑到与这些治疗相关的高毒性。在我们以前的工作中， 能够从局部晚期胰腺癌患者的血清中鉴定出20多种外泌体蛋白标志物， 通过使用外泌体分离方法和质谱分析，在治疗性治疗期间观察癌症。 然而，对于大的临床群组的这种分析受限于用于体外扩增的外泌体制备的再现性。 外泌体蛋白质的准确定量是外泌体组学研究领域的一个重要问题。 在目前的提案中，我们计划开发外泌体分离和表征的分析，重点是 严格性和可重复性我们将开发一种基于超级SILAC的（氨基稳定同位素标记） 细胞培养物中的酸）外泌体方法作为通用内标（UIS）。原则上，当超级SILAC- 将基于外泌体的蛋白质掺入人血清中进行蛋白质组学分析， 作为血清外泌体，其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标记的外泌体将是理想的，具有双特异性。 生物标志物分析功能，可重复样品制备并用作UIS。有针对性的LC-SRM和 来自我们先前研究的标记物的PRISM-SRM外泌体分析将与UIS一起使用，以精确监测 蛋白质在化学-放射治疗过程中的变化。局部晚期胰腺 癌症将在化疗治疗之前、期间和之后进行连续抽血， 化学辐射然后我们将进行基于外泌体标记的靶向蛋白质组学确认研究 在我们以前的工作中确定了对化学-放射治疗过程有特异性反应的细胞。标记 与基线相比的数值变化将与治疗结果（如无进展 生存期和总生存期）。这项工作将建立潜在的使用和测量 这些外泌体标志物的可重复性用于监测治疗期间和治疗后蛋白质标志物的变化。 本文所用的基于LC-SRM质谱的测定法具有独特的优点，即多路复用超过 20个标志同时该工作包括几个新的方面，包括：1）使用超- SILAC解决了外泌体蛋白准确定量的不重现性，特别是对于多步骤 外泌体制备和2）多重靶向LC-SRM和高灵敏度PRISM-SRM测定， 患者血清外泌体中的生物标志物分析。

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.