Abstract LB225: Toward comprehensive, single-molecule proteomics: Protein identification by short-epitope mapping

Abstract

Abstract Protein Identification by Short-epitope Mapping (PrISM) is a novel proteomics method with the goal of comprehensive proteome quantification at single-molecule sensitivity. In this work, we present computational modeling demonstrating the feasibility of this goal and experimental application of the approach leveraging single-molecule protein measurements to identify a small number of model proteins. The implementation of PrISM as presented works by acquiring sequential affinity reagent binding measurements on single protein molecules. Counterintuitively, using affinity reagents with “poor” specificity (i.e. cross-reacting to many proteins) enables identification of tens of thousands of proteins with only hundreds of affinity reagents. To experimentally demonstrate PrISM, we use a small number of affinity reagents to quantify a larger number of proteins. In simulations, PrISM is able to identify more than 98% of proteins in human, mouse, yeast, and E. coli using 300 affinity reagents targeting short trimer epitopes. PrISM is robust to experimental confounders including non-specific binding and noisy, incomplete, or inaccurate estimation of affinity reagent to protein binding characteristics. Simulations of the approach with a chip containing 10 billion protein molecules measured in parallel show a dynamic range of detection of up to 9.5 and 11.5 orders of magnitude for HeLa cells and plasma, respectively. Using such a chip, the PrISM method can identify and quantify over 90% of the human proteome in a single experiment, potentially revolutionizing biomarker discovery and cancer research. Citation Format: Jarrett D. Egertson, Cassandra Stawicki, Taryn E. Gillies, James H. Joly, Noah Steiner, Kevin Cardenas, Jonathan B. Leano, Kevin Chen, Sophie Watts, Hunter B. Boyce, Dan DiPasquo, Alana Killeen, Vadim Lobanov, Greg Kapp, Sujal Patel, Parag Mallick. Toward comprehensive, single-molecule proteomics: Protein identification by short-epitope mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB225.