Proteomic Profiling of RHD-Related Mitral Annulus Calcification Enabled by Magnetic Carbon Nanomaterial-Supported Quasi-Immobilized Enzyme Digestion.

Elucidating the mechanisms underlying rheumatic heart disease (RHD) accompanied by mitral annulus calcification (MAC) facilitates the identification of sensitive diagnostic biomarkers and the development of targeted therapeutic strategies. Proteomic analysis offers an approach to characterizing protein expression changes, thereby contributing to the clarification of pathogenesis in both RHD and cardiac valve calcification. Time-saving and high-efficiency enzymatic digestion methods are thus highly desirable for reliable and high-performance proteomic analysis. This study developed a quasi-immobilized enzyme digestion (QIED) technology based on honeycomb-like magnetic carbon material (Fe3C@C-650) with a pore size of 100-150 nm, which enables rapid enzymatic digestion in 5 min (200-fold faster than traditional methods) by ultrahigh enzyme adsorption capability. The developed method was applied to proteomic analysis of mitral valve tissues from 7 patients, and 3128 proteins were identified, which clarified the three-phase pathological progression (calcified, thickened, and normal tissues) of RHD-related calcification and revealed core hub genes (e.g., CXCL12, SRC, VCAM1) and the synergistic pathway of "inflammation regulation-protein translation-cell remodeling". This study provides methodological support and molecular targets for the precise diagnosis and targeted therapy of RHD complicated with MAC.