Identification of GOS Disaccharide Isomers Using cIMS-MS/MS

The characterization of isomeric oligosaccharides, such as galactooligosaccharides (GOS), in complex mixtures is a long-standing analytical challenge. Due to the exponential increase in the number of possible structures with an increasing degree of polymerization (DP), it is difficult to obtain a complete set of standards. Cyclic ion mobility mass spectrometry (cIMS-MS) has the potential to overcome this challenge by comparing oligosaccharide fragment ions with their respective disaccharide standards, thus reducing the reliance on higher DP oligosaccharide standards. To achieve this, effective separation and recognition of disaccharide fragments using cIMS is a primary prerequisite. Therefore, we studied the ability of combined cIMS separation and collision-induced dissociation (CID) fragmentation to identify isomeric disaccharides consisting of galactose (Gal) and glucose (Glc) with varying compositions and linkage types. Comparison of the drift time selectivity and fragment yields of lithium, sodium, potassium, rubidium, and cesium adducts revealed that lithium overall performed best, by providing good cIMS resolution and far superior fragment yields compared to the other metal ions. cIMS drift times were influenced primarily by the composition of disaccharides and consistently followed the order Gal-Gal < Gal-Glc < Glc-Glc for disaccharides with the same linkage type. CID cross-ring fragmentation patterns were linkage-type specific and did not strongly depend on composition or anomeric configuration. Overall, unknown disaccharides can straightforwardly be identified using our cIMS-MS/MS approach, as monosaccharide compositions can be deduced from cIMS drift times and linkage types from CID fragmentation patterns. These findings are a stepping stone for the identification of larger oligosaccharides by cIMS-MS-based approaches.