Juliane Merl-Pham (Neuherberg / DE), Manar Elkenani (Goettingen / DE), Fabian Gruhn (Neuherberg / DE), Stefanie M. Hauck (Neuherberg / DE), Karl Toischer (Goettingen / DE), Belal A. Mohamed (Goettingen / DE)
The cardiac extracellular matrix (ECM) plays a crucial role in providing structural, biochemical, and physiological support to the heart. Detecting ECM proteins through unbiased proteomic methods is challenging due to their solubility issues and relatively low abundance compared to the more prevalent cytoplasmic and mitochondrial proteins in the cardiomyocytes.
To delineate proteome-wide and ECM-specific proteome composition in wild type (C57BL/6N) murine hearts, we conducted label-free quantitative mass spectrometric analyses of both soluble proteins and ECM extracted from myocardial tissues. The heart tissue was mechanically lysed, sonicated to solubilize non-ECM proteins efficiently, and separated by centrifugation into soluble and insoluble fractions before undergoing tryptic digestion for subsequent analysis on a QExactive HF-X mass spectrometer. Data-independent acquisition was used for the soluble fraction, while data-dependent acquisition was used for the insoluble/ECM fraction. Protein identification and quantification were carried out using Spectronaut 18 (soluble fraction) or Proteome Discoverer 2.5 (insoluble/ECM fraction), followed by statistical analysis and data visualization in Perseus software.
In the soluble fraction we quantified over 4000 proteins, providing a detailed and comprehensive view of the molecular processes involved in the myocardial tissue. In the insoluble ECM fraction, we quantified more than 2000 proteins, including 155 known ECM proteins. This included 20 different collagen chains, 57 ECM glycoproteins, and 10 proteoglycans, providing a comprehensive understanding of the core matrisome in murine heart. The high-quality MSMS spectra also allowed for studying collagen chain stoichiometries and site-specific oxidations, giving insights into triple-helix structures and stability.
In conclusion, our study demonstrates the successful application of our established ECM enrichment protocol previously used on lung tissue to heart tissue. This comprehensive proteomics analysis contributes to our understanding of the physiological structure of the cardiac proteome and ECMome, offering a valuable resource for future studies on diseased hearts.