Abstract 5059: High-throughput monitoring of proteoforms and pathways through multiplexed and customizable mass spectrometry assay panels

Abstract

Abstract Proteins represent a key class of analytes in pharmacodynamic (PD) analysis, an essential component in preclinical and clinical studies which inform about a biological system’s response to drug treatment. Pharmacodynamic signatures typically comprise target engagement marker and downstream pathway markers. With recent development in targeted therapies and novel therapeutic modalities such as protein degraders, the scope of PD has widened to cover predictive and stratification biomarkers which can inform patient selection and contribute to further understanding of potential drug resistance. Using peptides as surrogate for proteins of interest, mass spectrometry (MS)-based approach alleviates the barriers of reagent specificity of antibody-based methods which can be affected by species and matrices used in preclinical drug development. Moreover, the use of stable-isotope labeled standard (SIS) reference peptides grants targeted MS assays unraveled specificity and the ability to determine absolute quantity of a given analyte. Harnessing the power of targeted MS, we present an oncology-focused assay repository which contains 804 peptides corresponding to 582 proteins. All 804 peptide surrogates were chosen under strict selection criteria such as uniqueness/proteotypicity and stability. Off-the-shelf availability (PQ500 kit) of SIS reference peptides for all 582 proteins facilitates the deployment of any of these assays in a plug-and-play manner. The collection of 582 protein targets cover > 180 cellular pathways (Reactome pathways e.g. cytokine signaling, cell-cell communication, activation of matrix metalloproteinases) together with 49 FDA-approved drug targets. To further evaluate the performance of these assays, we carried out a case control study comprising plasma samples from 5 patient groups (lung, colorectal, pancreatic, breast and prostate cancer) and age and gender-matched healthy subjects. In total, we measured absolute quantities of 582 proteins across 180 plasma samples while validating the expression profiles of well-characterized biomarkers in patient plasma. To conclude, we’ve demonstrated the application of targeted MS approach for the routine analysis of pharmacodynamic biomarkers. Its multiplexing capability, together with the adaptability to various species allow this method to maneuver through preclinical and clinical scenarios. Citation Format: Sebastian Müller, Véronique Laforte, Simonas Savickas, Maik Müller, Yuehan Feng, Lukas Reiter. High-throughput monitoring of proteoforms and pathways through multiplexed and customizable mass spectrometry assay panels. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5059.