RESUMO
Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/ß anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.