Optimized Proteomic and N -Glycoproteomic Profiling Reveals Aging-Associated Molecular Remodeling in Granulosa Cells

Granulosa cells (GCs) are essential for maintaining the follicular microenvironment and supporting oocyte development. However, the comprehensive profiling of the granulosa cell proteome and N-glycoproteome remains insufficiently characterized. Furthermore, the limited availability of clinical granulosa cell samples and the inherent difficulty in extracting membrane and secreted proteins necessitate a standardized sample preparation protocol. In this study, we systematically evaluated three protein extraction workflows (sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) and urea) alongside different digestion strategies (trypsin and tandem LysC-trypsin) for granulosa cell proteomic and N-glycoproteomic analyses. The SDS-based workflow showed superior overall performance and was applied to clinical granulosa cell samples from young (n = 6) and aged (n = 6) women. We successfully identified 7599 proteins and 4703 N-glycopeptides from the granulosa cell samples. Integrated proteomic and N-glycoproteomic analyses identified 12 differentially expressed proteins and 111 differentially abundant N-glycopeptides between young and aged GCs, suggesting alterations in protein expression and N-glycosylation associated with ovarian aging. Notably, changes in N-glycosylation were observed independently of changes in protein abundance. This study establishes a robust workflow for comprehensive proteomic and N-glycoproteomic profiling of clinical granulosa cell samples and provides novel insights into ovarian aging.