Next-Generation Oncoproteomic Sequencing and Precision Profiling

About This Grant

Project Summary Proteins are the essential workhorses of the cell, driving nearly every biological process. Their complex interactions, alterations, and post-translational modifications (PTMs) play critical roles in cancer development and progression. However, current protein analysis technologies suffer from significant limitations in sensitivity, accuracy, and throughput, hindering a comprehensive understanding of the proteomic landscape of cancer. There is an urgent need for a platform that can directly and accurately sequence individual proteins, identify variants, and profile complex modifications like phosphorylation and glycosylation, which are central to oncogenic processes. To address these limitations, we propose to develop an entirely new single-molecule protein sequencing platform capable of manipulating, controlling, and iteratively re-sequencing individual proteins. Our technology represents a transformative advance in oncoproteomics by offering direct, high-accuracy protein sequencing, overcoming the low throughput and labor-intensive nature of existing methods. By repeatedly sequencing the same protein molecule, our system provides "unlimited" coverage at the single-molecule level, enabling ultra-precise identification of protein variants and PTMs. This approach is essential for detecting single- point mutations and profiling cancer-related proteins with unprecedented accuracy. Our platform will leverage proven nanopore sequencing strategies adapted specifically for protein sequencing. The technology integrates multiple controllable methods to linearize proteins and control their movement through the nanopore, generating detailed current-time signatures for each amino acid. By controlling the translocation velocity and re-sequencing the same protein multiple times, we can construct high-accuracy consensus sequences, offering a gold-standard tool for oncoproteomic research. In this project, we will develop and optimize a 4-plex sequencing system capable of multiplexing measurements across independent nanopore sensors in a format that can be scaled to enable high-throughput sequencing in future efforts. This system will be validated using protein standards and then applied to sequence and profile the KRAS protein—a key oncogenic driver of cancer progression—and its mutated forms, along with relevant PTMs such as phosphorylation, glycosylation, and citrullination. Our goal is to demonstrate sequencing of cancer-relevant proteins with >98% accuracy. By enabling large-scale, high- throughput protein sequencing, this project will not only demonstrate its feasibility but also position it as a powerful tool for accelerating the discovery of new cancer biomarkers and therapeutic targets. The advancement of oncoproteomics through this novel technology holds the potential to revolutionize cancer diagnostics and treatment, offering more precise tools for early detection, disease monitoring, and personalized therapies.