Abstract 2338 Development of Methods for Identification and Quantification of Glycoconjugates

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Glycoconjugates are ubiquitous and of tremendous significance in biological systems. To understand their function, it is critical to be able to accurately elucidate their precise structure, even when only minute amounts can be obtained from cells or tissues. We will present new analytical tools for the analysis of glycosphingolipids (GSLs) and other glycoconjugates. GSLs are a group of glycolipids with a glycan head glycosidically linked to the C-1 hydroxyl group of a ceramide tail. While some diversity in GSLs is due to the ceramide group, the primary structural and functional categorizations are based upon glycan variability with impacts in cell adhesion, signaling, proliferation, endocytosis, intracellular transport, inflammation, and apoptosis. This study adopts a comparative approach for identification and quantification of GSLs. Therefore, two methods for analyzing GSLs were compared. In the first experiment, isolated GSLs were permethylated and analyzed using nanospray tandem mass spectrometry (nESI-MS/MS). In the second experiment, GSLs were first cleaved with endoglycoceramidase (EGCase) II, and the released glycans were fluorescently labeled and analyzed with UPLC-FLR-MS/MS. Notably, the UPLC approach led to greater resolution and higher sensitivity resulting from reduced heterogeneity by removing the ceramide. The EGCase II released glycans comprised both neutral and sialylated species of varying sizes. MS/MS was used for structure assignment confirmation in both methods. Since permethylation and procainamide labeling changes the m/z value of GSLs and glycoforms, the limited existing online databases and tools for lipids and metabolites are of small use for data interpretation. A database of possible m/z values for GSLs and glycoforms was compiled for assistance in data interpretation. Finally, we found that utilization of an internal standard can be helpful for quantification of GSLs. This work was supported by NIH R24GM137782, GlycoMIP, a National Science Foundation Materials Innovation Platform funded through Cooperative Agreement DMR1933525 and the the U.S. Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Division, under award #DE-SC0015662.