Studying Mucin-Derived O-Glycopeptides with Gas-Phase FRET.

At least 50% of human proteins are glycosylated, however, it is not fully understood how glycosylation affects glycopeptide structures. For the highly O-glycosylated tandem repeats of the gel-forming mucin MUC5AC, it has been shown that glycoclustering leads to significant stiffening of the peptide backbone in solution. Here, the influence of O-glycosylation on the gas-phase structures of synthetic 26-residue MUC5AC-derived model peptides containing either zero or six GalNAc residues is investigated using gas-phase Förster resonance energy transfer (FRET) and ion mobility-mass spectrometry. The results reveal that O-glycosylation can induce pronounced compaction after desolvation, in contrast to the glycosylation-induced stiffening in solution. The extent of the compaction is charge state-dependent and likely related to intramolecular solvation of the glycan residues. Additionally, the results show that glycopeptides with Thr-glycosylation are either less compact or of similar size than those with Ser-glycosylation. These results highlight the importance of hydration for stabilizing glycopeptide conformations and show that glycosylation-dependent structural changes should be considered when interpreting gas-phase measurements of glycopeptides.