Compartment-Resolved Proteomics with Deep Extracellular Matrix Coverage.

The extracellular matrix (ECM) is a complex network of proteins that provides structural support and biochemical cues to cells within tissues. Characterizing ECM composition is critical for understanding this tissue component's roles in development, homeostasis, and disease processes. This protocol describes an integrated pipeline for profiling both cellular and ECM proteins across varied tissue types using mass spectrometry-based proteomics. The workflow covers stepwise extraction of cellular and extracellular proteins, enzymatic digestion into peptides, peptide cleanup, mass spectrometry analysis, and bioinformatic data processing. The key advantages include unbiased coverage of cellular, ECM-associated, and core-ECM proteins, including the fraction of ECM that cannot be solubilized using strong chaotropic agents such as urea or guanidine hydrochloride. Additionally, the method has been optimized for reproducible ECM enrichment and quantification across diverse tissue samples. This protocol enables systematic mapping of the ECM at a proteome-wide scale. Key features • Improved profiling of core extracellular matrix and matrisome-associated proteins through multi-step decellularization and chemical extraction of insoluble ECM • Extraction buffers optimized for effectiveness across a broad range of tissue types and compatibility with varied MS platforms • Measurement of protein solubility via resistance to detergent and chaotrope extraction • Integrated LC-MS/MS analysis and data processing pipeline for ECM-focused analysis.