Metabolic labeling of glycans with isotopic glucose for quantitative glycomics in yeast.

ABSTRACT

Changes in glycan levels could directly affect the biochemical properties of glycoproteins and thus influence their physiological functions. In order to decode the correlation of glycan prevalence with their physiological contribution, many mass spectrometry (MS) and stable isotope labeling-based methods have been developed for the relative quantification of glycans. In this study, we expand the quantitative glycomic toolbox with the addition of optimized Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) approach in baker's yeast (Saccharomyces cerevisiae). We demonstrate that culturing baker's yeast in the presence of carbon-13 labeled glucose (1-13C1) leads to effective incorporation of carbon-13 to both N-linked and O-linked glycans. We established that metabolic incorporation of isotope-labeled glucose at a concentration of 5 mg/mL for three days is required for an accurate quantitative analysis with optimal isotopic cluster distribution of glycans. To validate the robustness of the method, we performed the analysis by 11 mixing of normal and isotope-labeled glycans, and obtained excellent linear calibration curves from various analytes. Finally, we quantitated the inhibitory effect of tunicamycin, a N-linked glycosylation inhibitor, to glycan expression profile in yeast.