Sialylation-induced stabilization of dynamic glycoprotein conformations unveiled by time-aligned parallel unfolding...

Sialylation, a critical post-translational modification, regulates glycoprotein structure and function by tuning their molecular heterogeneity. However, characterizing its subtle and dynamic conformational effects at the intact glycoprotein level remains challenging. We introduce a glycoform-resolved unfolding approach based on a high-throughput ion mobility-mass spectrometry (IM-MS) platform. This method integrates high-throughput unfolding with parallel fragmentation, enabling simultaneous analysis of sialylation patterns, stoichiometries, and their impact on conformational stability. Applying this approach to fetuin, we identified distinct sialylation patterns and their differential influence on protein conformation, namely sialylation-induced stabilization during early unfolding and increased flexibility in later unfolding stages. IM-MS-guided molecular dynamics simulations revealed that increased sialylation enhances the initial conformational stability, likely through enhanced electrostatic interactions and hydrogen bonding. These findings highlight the complex interplay between sialylation and protein dynamics and establish glycoform-resolved unfolding IM-MS as a powerful tool for characterizing glycoprotein conformational landscapes.