Determining β-Monosaccharide Head Group Composition with High-Resolution Cyclic Ion Mobility Separations Coupled to Tandem Mass Spectrometry as a First Step for Unknown Cerebroside Analysis.

Cerebrosides, a class of biologically important lipids, are comprised of a monosaccharide head group along with their ceramide tail. However, their accurate characterization is challenging because of the isomerism in both the tail, from potential double bond positioning, or in the head from monosaccharide composition and αβ anomericity. In this work, we focused on tackling the identification of the β-monosaccharide head group, as either glucose or galactose, in various cerebroside isomers as well as demonstrating how our methodology could be applied to unknowns found in a porcine extract. To achieve this, we performed collision-induced dissociation prior to cyclic ion mobility separations to generate monosaccharide fragment ions from the starting cerebroside precursor ions. With this pre-cIMS CID approach, we observed that the cIMS separations of the fragment ions were diagnostic of the β-monosaccharide head group composition (i.e., glucose versus galactose), regardless of the ceramide tail length. From there, we demonstrated an example of how this methodology could also be applied to cerebrosides found in a porcine extract and a framework for how this approach could be added to existing workflows in developing collision cross section databases. Overall, we envision that our developed pre-cIMS CID-based approach will be a complementary and orthogonal tool to existing ones in glycolipidomics workflows.