Possemato, A. P. | bioRxiv Science

bioRxiv2025

Expanding the Global Map of Protein Post Translational Modifications with Immunoaffinity Enrichment and nDIA Analysis on the Orbitrap Astral Mass Spectrometer

Post-translational modifications (PTMs) contribute greatly to the diversity of the human proteome by affecting protein structure, function, interactions, stability, localization, and more. The study of PTMs is essential to understand various cellular functions, disease mechanisms, and aid in the development of biomarkers and design of therapeutic targets. Owing to their diversity, dynamic nature, and low stoichiometry compared to unmodified proteome counterparts, the analysis of PTMs remains challenging. In this study, immunoaffinity enrichment of PTM peptides was combined with analysis using Data Dependent Acquisition (DDA) and narrow window Data Independent Acquisition (nDIA) on the Orbitrap Astral mass spectrometer (MS) as well as comparative analysis using the Orbitrap Fusion Lumos MS for Ubiquitination, Phosphorylation, Acetylation, Succinylation, and Methylation. Human cell line and mouse tissue samples at various input peptide amounts were immuno-enriched and mass spectrometry data was acquired on both instruments to assess depth of coverage and number of novel sites identified. The study identified a total of 88,731 unique ubiquitin sites, 64,397 phosphorylation sites (43,721 phosphoserine, 8,414 phosphothreonine and 12,262 phosphotyrosine), 11,629 acetylation, 5,272 succinylation and 1,461 mono-methylation sites. In half the acquisition time, nDIA analysis of immuno-enriched samples on Orbitrap Astral MS provided much greater depth of coverage for all PTMs compared to DDA analysis on Orbitrap Fusion Lumos MS, with up to 33-fold more PTM peptides identified and quantified. Overall, the data presented in this study demonstrates the need for enrichment for PTM detection and the utility of combining antibody-based peptide capture and nDIA on the Orbitrap Astral MS as powerful tools for discovery and profiling of protein post-translational modifications in cells and tissues.

biochemistry↗

bioRxiv2023

Identification of chemotherapy targets reveals a nucleus-to-mitochondria ROS sensing pathway

Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, its unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H2O2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H2O2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.

molecular biology↗