Simultaneous quantification of nucleotide sugar metabolism by LC and LC-MS

Cellular glycosylation plays an important role in many biological processes, as well as in disease states such as diabetes and cancer. Nucleotide sugars are donor substrates of glycosyltransferases and their availability and localization regulate glycosylation status. The nucleotide sugar level is controlled by cellular metabolic states. To investigate the fate of nucleotide sugars in glycosylation, two methods for monitoring nucleotide sugar metabolism, namely, ion-pair reversed-phase HPLC and LC-MS, have been previously described (Nakajima et al., Glycobiology 20 (7):865–871, 2010; Mol Cell Proteomics (9):2468–2480, 2013). Using the HPLC method, cellular levels of eight unique nucleotide sugars were simultaneously determined. Using the LC-MS method, which is based on mass isotopomer analysis of nucleotide sugars metabolically labeled with¹³C6-glucose, the metabolic pathways governing UDP-GlcNAc synthesis and utilization were characterized. Here, these strategies are reviewed and the biochemical significance of nucleotide sugars in cellular glycosylation is discussed.