Ji Eun Park (Chungnam National University)

Hyun Joo An (Chungnam National University)

Ji Eun Park (Chungnam National University)

Myung Jin Oh (Chungnam National University)

Hyun Joo An (Chungnam National University)

Glycosphingolipids (GSLs) abundantly present on cell surfaces play pivotal roles in cell-cell communication, immune response, and recognition. The biological functions of GSLs depend intricately on the glycan and ceramide backbone, comprehending their intact structure to elucidate physiological roles is imperative. However, the amphipathic nature of GSLs, with hydrophilic glycans and hydrophobic ceramides, poses challenges to their analysis. Furthermore, based on the glycan head structure, it exhibits neutral and acidic chemical properties. In this study, we developed an MS-based analytical platform for simultaneous profiling neutral and acidic GSLs. GSL isomers were effectively separated on a C18 column. This allowed ‘RT prediction’ by constructing of a library based on the elution behavior of GSLs on C18 columns. The library, constructed using a mixture of GSLs with known structures and composition, facilitated the prediction of retention times based on the experimentally determined correlation between  retention time and the total carbon number of ceramide. Subsequent confirmation of the separated GSLs was achieved by MS/MS fragmentation. We identified not only isomers of representative acidic GSLs such as GD1a and GD1b but also isomers with different ceramide and sialic acid species(NeuAc, NeuGc). Additionally, the isomers of ceramide(Gb4 40:2;O2) separated on C18 column, were identified through diagnostic fragment ions of the sphingosine. This platform was successfully applied to various biological samples, identifying cell- and organ-specific GSLs.

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