Universal Internal Standard for Reproducible Accurate Quantification of Exosome Protein Markers

摘要：胰腺癌目前是美国与死亡相关的第三大癌症原因，占美国癌症死亡人数的5%。 一年存活率为5%。大多数潜在治愈的局部晚期不能切除的患者 治疗标准包括化疗和放射治疗相结合的疾病。一位少校 胰腺癌治疗面临的挑战是早期评估治疗反应。小说 迫切需要早期治疗反应的标记物，以便更好地做出有关类型的知情决定 以及治疗的顺序，考虑到与这些治疗相关的高毒性。在我们之前的工作中，我们 我们能够从局部晚期胰腺癌患者的血清中识别出20+个胞外蛋白标记物 肿瘤治疗过程中采用外切体分离方法和质谱分析。 然而，这种对大型临床队列的分析受到外切体制剂重复性的限制。 外体蛋白的准确定量是外体组学研究领域的一个重要问题。 在目前的提案中，我们计划重点发展外切体分离和特征的分析 关于严格性和可重复性。我们将开发一种超SILAC为基础的(稳定的同位素标记/与氨基 细胞培养中的酸)外切体方法作为通用内标(UIS)。原则上，当超SILAC- 将基于外切体的蛋白质添加到人血清中进行蛋白质组分析，它可以遵循相同的实验流程 作为血清外切体，可以最大限度地减少样品制备和LC-MS带来的变异 (液相色谱-质谱仪)分析。因此，SILAC标记的外切体将是理想的具有双重 生物标记物分析的功能，用于可重复的样品制备，并充当UIS。有针对性的LC-SRM和 我们以前研究的标记的PRISM-SRM外显体分析将与UIS一起使用，以精确监测 放化疗过程中蛋白质的变化。局部晚期胰腺癌患者 癌症患者将在化疗前、化疗中和化疗后连续抽血，然后 化疗放射治疗。然后，我们将根据外显子标记进行有针对性的蛋白质组学确认研究 在我们之前的工作中发现了对一个疗程的化疗-放射治疗有特定反应的药物。记号笔 与基线相比的值变化将与治疗结果(如无进展)相关 存活率和总存活率)。这项工作将建立潜在的用途和测量 这些胞外体标记物用于监测治疗期间和治疗后蛋白质标记物变化的重复性。 在此使用的LC-SRM质谱学分析的明显优点是可以被多路复用 同时有20个记号笔。拟议的工作包含几个新颖的方面：1)使用超级 SILAC将解决外显体蛋白准确定量的不重复性问题，特别是多步骤 外切体制备和2)多重靶向LC-SRM和高灵敏PRISM-SRM分析 患者血清外切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.