Manipulation of Ion Types via Gas-Phase Ion/Ion Chemistry for the Structural Characterization of the Glycan Moiety on Gangliosides.

Gangliosides are the most abundant glycolipid among eukaryotic cell membranes and consist of a glycan head moiety containing one or more sialic acids and a ceramide chain. The analysis of the glycan moieties among different subclass gangliosides, including GM, GD, and GT gangliosides, remains a challenge for shotgun lipidomics. Here, we present a novel shotgun lipidomics approach employing gas-phase ion/ion chemistry. The gas-phase derivatization strategy provides a rapid way to manipulate the ion-types of the precursor ions, and, in conjunction with collision induced dissociation (CID), allows for the elucidation of the structures of the glycan moieties from gangliosides. In addition to the enhancement of structural characterization, gas-phase ion chemistry leads to a form of purification of the precursor ions prior to CID by neutralizing isobaric or isomeric ions with different charge states but with similar or identical m/z values. To demonstrate the proposed strategy, both deprotonated GM3 and GM1 gangliosides ([GM-H](-)) were isolated and subjected to reaction with magnesium-Terpy complex cations ([Mg(Terpy)(2)](2+)). The post-reaction product spectra show the elimination of possible contamination, illustrating the ability of charge-switching derivatization to purify the precursor ions. Isomeric differentiation between GD1a and GD1b was achieved by the sequential ion/ion reactions, with the CID of [GD1-H+Mg](+) showing diagnostic fragment ions from the isomers. Moreover, isomeric identification among GT1a, GT1b, and GT1c was accomplished while performing a gas-phase magnesium transfer reaction and CID. Lastly, the presented workflow was applied to ganglioside profiling in a porcine brain extract. In total, 34 gangliosides were profiled among only 20 precursor ion m/z values by resolving isomers. Furthermore, the fucosylation site on GM1 and GD1, and N-glycolylneuraminic acid conjugated GT1 isomers was identified. Relative quantification of isomeric two isomeric pairs, GD1a/b C36:1 and GD1a/b C38:1 was also achieved using pure component product ion spectra coupled with a total least-squares method. The results demonstrate the applicability and strength of using shotgun MS coupled with gas-phase ion/ion chemistry to characterize the glycan moiety structures on different subclasses of gangliosides.