Applications of the IDAWG technique to quantitative glycomics of human embryonic stem cells

Abstract

As the first reported in-vivo stable isotopic labeling strategies for quantitative glycomics, the IDAWG (Isotopic Detection of Aminosugars With Glutamin) takes advantage of the hexosamine biosynthetic pathway that provides the nitrogen to all aminosugars from the side chain of glutamine (Gln). As a result, if the cells are fed with Gln-free media and glutamine with a 15N-labeled side chain is added, all the aminosugars produced in cells will be labeled with 15N and thus the mass of all glycan structures will be shifted by approximately +1 dalton per aminosugar. In this work, we demonstrate the utility of the IDAWG technique to study both static and dynamic glycomics of human embryonic stem cells. To compare N-linked and O-linked glycan expression levels in hESCs and hDE and to quantify the changes in glycan expression that accompany differentiation of hESCs into hDEs, four cell populations including light and heavy hESCs, light and heavy hDEs have been processed and N- and O-linked glycans have been released. After the permethylated glycan samples were analyzed by ESI-LTQ-Orbitrap, more than 20 O-linked glycan structures and 90 N-linked glycan structures were assigned in hESCs and hDEs. Further, these glycans were quantified to show the prevalence of each structure in the total glycan profiles. Based on the mass spectrometry data of mixed samples of hES and hDE, glycan expression level changes were quantified for a subset of the glycans. To apply the IDAWG technique to assess the dynamics of turnover and synthesis for individual glycans and to provide a dynamic view of the glycome, a pulse-chase labeling of hESCs was performed. Heavy labeled cells were shifted to light media for 0, 6, 12, 24 or 36 hr before harvesting, isolation and permethylation. Analysis of these samples allowed us to approximate the turnover and synthesis rate of the glycans. Interestingly, by comparing the glycan spectra of pulse-chase samples with the spectra of real mixtures of light and heavy sample, we noticed an isotope mixture that could only be explained by remodeling of the glycans and were able to approximate the amount of remodeling occurring for a subset of structures.