Advanced N-glycoproteomics and proteomics approach revealed sexually dimorphic molecular signatures in primary mouse hepatocyte.

Sexual dimorphism plays a critical role in disease pathophysiology, but the subtlety and complexity of these differences, along with a lack of precise comparative methods, hinder the advancement of precision medicine and drug development. This limitation is particularly evident in metabolic dysfunction-associated steatotic liver disease (MASLD), where sex-specific molecular mechanisms remain insufficiently understood. To address this gap, we employed an advanced integrative N-glycoproteomics and proteomics approach to systematically analyze sex-biased molecular signatures in primary mouse hepatocytes (PMHs) under healthy and MASLD conditions. Our analysis identified 280 sex-biased proteins and 39 sex-biased N-glycosites, and KEGG enrichment revealed that female-biased molecules were primarily involved in lipid metabolism, while male-biased molecules were associated with inflammation and cytoskeletal remodeling. A combined dataset of 302 sex-biased molecules was further analyzed using protein-protein interaction (PPI) analysis and Rc value calculations, resulting in the identification of 21 hub proteins and 2 hub N-glycosites as MASLD-associated sex-biased signatures. Notably, MASLD amplified proteomic sex differences while attenuating them in N-glycosylation. Western blot validation of key signatures, including female-biased MVK and male-biased LGALS3, highlighted distinct molecular adaptations between the sexes in MASLD progression. Our study introduced an advanced analytical framework for high-resolution comparative molecular profiling by integrating N-glycoproteomics with proteomics, providing valuable insights into sex-biased molecular signatures, enhancing preclinical model development, and advancing sex-specific therapeutic strategies in MASLD research and broader biological systems.