SDD-AGE-MS · SDD-AGE-MS: a proteomics tool for characterizing protein organization and plasticity in biomolecular condensates.

In living systems, biological processes are orchestrated by proteins organized into dynamic assemblies. Cells regulate protein assembly and disassembly through various mechanisms, with subcellular compartmentalization playing a critical role in creating a microenvironment that control biological reactions. Notably, not all compartments rely on membranes for segregation; biomolecular condensates, for example, are membraneless organelles formed through a network of multivalent interactions. While noteworthy progress has been made in identifying the proteins partitioning in biomolecular condensates and understanding the forces driving phase separation; how proteins interact forming assemblies within condensates remain poorly understood. This knowledge gap is primarily due to the lack of system-wide approaches capable of effectively characterizing condensates, which are often low in abundance, large, dynamic, and highly heterogeneous.In this study, we introduce SDD-AGE-MS, an innovative proteomics method designed to characterize the architecture of condensates. By integrating SDD-AGE, an electrophoretic technique commonly used to visualize SDS-resistant high molecular weight assemblies, with mass spectrometry (MS), we aim to map the migration profiles of SDS-resistant proteins and link them to protein assemblies.This project will make significant contributions on multiple levels: (i) conceptually, by unveiling and resolving sub-assembly organization of condensates; (ii) methodologically, by introducing a system-wide approach for characterizing high molecular weight assemblies in cells; and (iii) translationally, by providing insights into the mechanisms driving condensate formation and their transition to pathological aggregates, as observed in neurodegenerative diseases.