IDAWG: Metabolic Incorporation of Stable Isotope Labels for Quantitative Glycomics of Cultured Cells

Abstract

Robust quantification is an essential component of comparative –omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-15N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 hours in the presence of amide-15N-Gln media results in nearly complete incorporation of 15N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox.

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

“…3) Because LC-MS analysis can roughly separate complex cellular glycans, this method enabled us to evaluate the labeling efficiencies of each isometric glycan separated on a graphite carbon column. The directly infused approach that relies on permethylated glycans metabolically labeled with 15 N-glutamate in the hexosamine pathway has been reported previously (17). However, tracing the dynamics of isometric glycans is not ap- proachable by a directly infused approach.…”

“…It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.…”

Quantitative Liquid Chromatography-Mass Spectrometry-Multiple Reaction Monitoring (LC-MS-MRM) Analysis of Site-specific Glycoforms of Haptoglobin in Liver Disease

“…Improvements in the mass spectrometric quantification of permethylated glycans based on the introduction of D or 13C isotopic labels were proposed but have not yet reached wide acceptance (24 -26). Elegant isotopic labeling of glycans in cell culture, similar to SILAC labeling of peptides, was also developed but remains to be used more widely (24,26). Methods for relative quantification of detached glycans based on the labeling of the reducing end or sialic acids represent further efforts to improve mass spectrometric quantification of detached N-glycans in this fast growing field of science (11,(27)(28)(29)(30).…”

Quantitative Glycomics Strategies

“…This was recently implemented in a metabolic labeling strategy known as the isotopic detection of amino sugars with glutamine (75). When amide-15 N-Gln media is used to culture cells, 15 N is incorporated into glycans (Fig.…”