Confident identification of isomeric N-glycan structures by combined ion mobility mass spectrometry and hydrophilic interaction liquid chromatography.

Rationale: A central issue in glycan mass analysis is the ambiguity of structural assignments due to the heterogeneity and complexity of glycan structures. Ion mobility mass spectrometry (IM-MS) has the potential to separate isomeric glycans depending on their unique collisional cross section especially when coupled with hydrophilic interaction liquid chromatography (HILIC).

Methods: Ten pyridylaminated biantennary N-glycans including isomeric structures were measured by electrospray ionization quadrupole-time-of-flight mass spectrometry with an ion mobility phase. We investigated which adduct ions would be suitable for good separation in the ion mobility phase. The differences in observed drift time of isomeric glycans were assessed by molecular dynamics (MD) simulations in vacuum. Connecting an HILIC system with IM-MS provided another, augmented separation mode.

Results: By selecting doubly protonated precursor ion species, we succeeded in separating a pair of isomeric glycans in the ion mobility phase with reasonable resolution. MD simulations of monogalactosylated glycan isomers indicate that the galactosylated Man α1-3 branch preferentially folds back to the core chitobiose portion to form a compact structure. IM-MS combined with HILIC resulted in even clearer separation of isomeric glycans within 15 min.

Conclusions: A combination of IM-MS with an HILIC system is eminently suitable for the confident and rapid distinction of glycan structures within a defined mixture.