Untargeted Serum Proteomics in the Fontan Circulation Reveals Three Distinct Molecular Signatures of Fontan Physiology with CYB5R3 Among Key Proteins

The total cavopulmonary anastomosis (Fontan procedure), a palliative procedure for single-ventricle congenital heart disease, improves survival but is associated with progressive multiorgan complications and high long-term morbidity. Prior blood-based proteomic studies in adults have been limited to targeted antibody-based panels or focused on methodological comparisons. Systemic molecular alterations in younger, clinically heterogeneous patients, particularly in untargeted pathways, remain incompletely characterized. Serum samples from 48 Fontan patients and 48 age- and sex-matched healthy controls were analyzed using mass spectrometry with TMT labeling. 2228 proteins were quantified, of which 124 were significantly differentially abundant (fold change > 1.5 or <0.67, FDR-adjusted <i>p</i> < 0.05). Network analysis identified three major functional clusters: extracellular matrix (ECM) organization (predominantly increased), actin cytoskeleton organization, and platelet-related pathways (both predominantly decreased). Stratified analyses showed reduced ECM protein abundance in high-risk patients, suggesting a shift from active remodeling toward a more established fibrotic state, and uniquely elevated cytochrome b5 reductase 3 (CYB5R3), implicating altered redox homeostasis, nitric oxide metabolism, and cellular aging. Overall, our findings extend prior targeted analyses, reveal potential biomarkers such as CYB5R3 and underscore the complexity of the Fontan circulation, with implications for risk stratification and therapeutic targeting.