In situ global mapping of protein perturbations via protein abundance and conformation analysis.

Background: Traditional studies of protein responses to external stimuli primarily focus on changes in protein abundance, often overlooking the critical role of protein conformational alterations. To address this gap, we developed Protein Abundance and Conformation Analysis (PACA), an integrative method that quantifies both protein abundance and conformational changes. PACA combines conventional quantitative proteomics for abundance measurements with Target Response Accessibility Profiling (TRAP), a technique that captures conformational changes in situ by applying reductive dimethylation to label accessible lysine residues in living cells before lysis.

Results: To address the need to quantify both protein abundance and conformational changes, we classified the proteome into four categories: Abundance Responders (AR), with abundance changes alone; Structural Responders (SR), showing conformational changes only; Co-Responders (CoR), exhibiting both conformational and abundance changes; and Non-Responders (NR), with no detectable changes. Using a lipopolysaccharide (LPS)-induced microglial activation model, PACA identified 49 AR proteins, 82 SR proteins, 4 CoR proteins, and 4045 NR proteins. AR proteins, including inflammatory mediators like cytokines, were critical for initiating inflammatory responses. SR proteins were enriched in processes such as energy metabolism, protein folding, and translation. Notably, four CoRs associated with inflammatory responses were detected: ACOD1, JunB, CCL4, and GPR84. By integrating both protein abundance and structural data, PACA provides comprehensive insights into the dynamics of microglial activation and immune regulation.

Significance: This method provides a robust framework for mapping proteome-wide responses by integrating protein abundance and structural analyses. This dual-focus approach uncovers critical insights into protein dynamics and their roles in cellular processes. By revealing regulatory proteins and pathways involved in microglial activation, PACA advances our understanding of immune responses, providing a valuable tool for exploring complex biological mechanisms across diverse conditions.