Targeted proteomics of MUC16 to enable early detection of ovarian cancer recurrence

高级别浆液性卵巢癌（HGSOC）是一种致命的疾病，在很大程度上是因为大多数病例复发， 在这一点之后，几乎没有什么可以做的。迫切需要新的诊断工具来改善HGSOC管理 并在临床表现之前检测复发。MUC 16在卵巢癌细胞上过表达， 目前在临床检测中检测到的CA 125表位。CA 125滴度的监测价值是 在妇科肿瘤学领域的积极辩论。一些研究报告没有生存益处， 而另一些人则指出，如果迅速采取行动，可以进行更高质量的二次细胞减灭术。成功 对抗HGSOC需要开发新的检测方法，以更可靠和灵敏地检测复发性HGSOC。 疾病我们的目标是通过从根本上重新思考MUC 16来开发HGSOC复发的新生物标志物。 我们认识到MUC 16本身存在，而不是将MUC 16视为CA 125表位的载体 作为多种多样的蛋白形式。由于mRNA剪接、翻译后切割和翻译后切割的变化， 由于存在翻译修饰，个体中存在的MUC 16分子群体是异质的。 MUC 16是大量糖基化的，并且来自癌细胞的MUC 16的聚糖谱是未开发的。 丰富的诊断信息，现在使用现有的基于肽的CA 125免疫测定丢失。的 该项目的目标是开发一种方法，从接受治疗的妇女血清中富集MUC 16， HGSOC并使用靶向（糖）蛋白质组学分析MUC 16。我们假设， 使用现代生物分析和计算工具揭示的MUC 16将使新的诊断能够检测 HGSOC的复苏比目前可能的要早。这一假设将通过以下三个方面进行研究： 研究目的。在目标1中（开发使用质谱定量MUC 16（糖）肽文库的方法）， 为了开发一种优化的免疫荧光光谱法（例如，免疫荧光光谱法），来自HGSOC患者的库存腹膜液的MUC 16将用于开发优化的免疫荧光光谱法。 用于鉴定MUC 16糖肽和肽之前和之后的新型靶向质谱法 去糖基化后。将通过检查下列研究生成平行反应监测（PRM）入选列表： silico digest数据和实验MS/MS数据。目标2（开发一种微量分离方法， MUC 16来自血清），我们将采用我们开发的无免疫亲和性的方案来富集MUC 16， 腹膜液中分离粘蛋白从血清样品。将使用一种基于数据库的格式来捕获 MUC 16基于其负电荷和特异性糖基化。最后，在目标3中：（编制关于以下方面的试点数据： 治疗前和治疗期间患有HGSOC的患者中MUC 16（糖）肽文库的纵向变化）， 来自HGSOC患者的纵向血清样品将使用微量方案富集MUC 16。 将对样本进行蛋白水解，并通过nLC-PRM进行分析。MUC 16的定量（糖）肽图谱，来自 将纵向比较每个患者。样品之间偏离最大的（糖）肽将是 在未来的研究中确定为早期检测复发HGSOC的潜在诊断标志物。

High-grade serous ovarian cancer (HGSOC) is a deadly disease, in large part because most cases recur, and little can be done after that point. New diagnostic tools are urgently needed to improve HGSOC management and detect recurrence before clinical presentation. MUC16 is overexpressed on ovarian cancer cells and bears the CA125 epitope that is currently detected in clinical assays. The value of CA125 titers for surveillance is an active area of debate within the gynecologic oncology community. Some studies report no survival benefit, while others point to higher quality secondary cytoreductive surgery if action is taken quickly. Successfully fighting HGSOC requires that new assays are developed for more reliable and sensitive detection of recurrent disease. Our goal is to develop a new biomarker for HGSOC recurrence by fundamentally rethinking MUC16. Instead of considering MUC16 as the carrier of the CA125 epitope, we recognize that MUC16 itself is present as multiple diverse proteoforms. Because of variation in mRNA splicing, post-translational cleavage, and post- translational modifications, the population of MUC16 molecules present in an individual is heterogeneous. MUC16 is abundantly glycosylated, and glycan profiles of MUC16 derived from cancer cells is an untapped wealth of diagnostic information that is now lost using the existing peptide-based CA125 immunoassay. The goal of this project is to develop a method to enrich MUC16 from the serum of women undergoing treatment for HGSOC and analyze MUC16 using targeted (glyco)proteomics. We hypothesize that the molecular diversity of MUC16 revealed using modern bioanalytical and computational tools will enable novel diagnostics to detect HGSOC resurgence earlier than is currently possible. This hypothesis will be investigated by pursuing three research aims. In Aim 1 (Develop methods to quantitate MUC16 (glyco)peptide libraries using mass spectrometry), MUC16 from banked peritoneal fluid of HGSOC patients will be used to develop an optimized novel targeted mass spectrometry method for identification of MUC16 glycopeptides and peptides before and after deglycosylation. A parallel reaction monitoring (PRM) inclusion list will be generated by examination of in silico digest data and experimental MS/MS data. In Aim 2 (Develop a microscale separation method to enrich MUC16 from serum), we will adapt an immunoaffinity-free protocol that we developed to enrich MUC16 from peritoneal fluid to isolate the mucin from serum samples. A cartridge-based format will be used to capture MUC16 based on its negative charge and specific glycosylation. Finally, in Aim 3: (Develop pilot data on longitudinal variations in MUC16 (glyco)peptide libraries in patients with HGSOC before and during treatment), longitudinal serum samples from HGSOC patients will be enriched for MUC16 using the microscale protocol. Samples will be proteolyzed and analyzed by nLC-PRM. Quantitative (glyco)peptide maps for MUC16 from each patient will be compared longitudinally. The (glyco)peptides that deviate most between samples will be determined as potential diagnostic markers for early detection of recurring HGSOC in a future study.