Development of Personalized Intact Proteome Map for Ovarian Cancer Patients Using Ultrasensitive Multiplexed Top-down Proteomics

Development of Personalized Intact Proteoform Map for Patient-derived Ovarian Cancer Spheroids Using Ultrasensitive Multiplexed Top-down Proteomics Project Summary Ovarian cancer is currently the deadliest gynecological malignancy; however, treatment still largely relies on untargeted chemotherapies that often lead to chemoresistant recurrence. Unlike many cancers that spread through the blood, ovarian cancer metastasizes through multicellular cancer spheroids that form in ascitic fluid. It has been reported that chemoresistance in ovarian cancer is mediated by chemoresistance in the spheroids in the ascites. Moreover, protein post-translational modifications (PTMs) in spheroids, such as phosphorylation, have been reported to play a critical role in ovarian cancer metastasis, treatment response, and chemoresistance. Therefore, analyzing proteins and their PTMs in individual spheroids isolated from patient ascitic fluid holds great potential for the discovery of disease biomarkers, early detection of chemoresistance, and development of personalized and effective treatments. Top-down proteomics of individual ovarian cancer spheroids could reveal key disease biomarkers and guide personalized treatments, but achieving this requires developing new ultrasensitive methods. The overall objective of this proposal is to develop an integrated top- down proteomics and phosphoproteomics platform to generate personalized intact proteome maps from patient- derived ovarian cancer spheroids—including those undergoing chemotherapy—to characterize the disease states and identify markers of chemoresistance. We recently developed and patented a novel ‘Spray-Capillary’ technique for ultralow volume sampling (nL or pL injection volumes). We recently developed and patented a novel ‘Spray-Capillary’ technique for ultra-sensitive (<100 pg) top-down capillary electrophoresis mass spectrometry (CE-MS) analysis. Here, we propose the integration of this platform with our long capillary ultrahigh pressure UPLC strategy for deep top-down proteomics. We propose to achieve this goal through two specific aims: (Aim 1) developing and benchmarking a 2D-RPLC-CE-MS/MS platform for global intact proteoform screening for spheroids; and (Aim 2) developing and benchmarking a 3D-IMAC-RPLC-CE-MS/MS platform for low abundance intact phosphoproteoform screening for spheroids. The proposed platform is highly innovative and sensitive, allowing the formation of personalized proteoform maps from single spheroids collected directly from patient ascites. This technology provides deeper insights into the development of ovarian cancer and facilitates personalized clinical treatment. Moreover, this transformative approach holds great promise for studying other mass-limited clinical samples in cancer research, such as biopsy samples or even single cancer cells, providing unique top-down MS data on healthy and cancerous human tissues that exceeds current capabilities.