O-glycosylation is an essential post-translational modification, controlling biological processes like innate immunity, lubrication, and cell communication. Due to the inherent structural complexity of O-glycans, assigning exact structures by mass spectrometry is highly challenging. Detailed structural assignment involves laborious processing and data interpretation but often remains incomplete due to the absence of diagnostic fragments. In this work, we introduce cryogenic gas-phase infrared spectroscopy to facilitate the characterization of O-glycans. We show O-glycans provide highly diagnostic IR fingerprints, which allow us to easily distinguish between isomeric O-glycan core structures. Ab initio calculations are used to understand the gas-phase structures of the deprotonated O-glycans. The experimental spectra suggest contributions from distinct ensembles of deprotomers and conformers present at cryogenic temperatures in the gas phase that are not detected in ion mobility spectrometry experiments at ambient temperature. We find that rapid charge migration processes, reminiscent of the Grotthuss mechanism in water, allows interconversion of deprotomers at ambient temperatures due to the presence of dense hydrogen-bond networks and the high flexibility of O-glycans in the gas phase.