Glycosaminoglycanomics of Cultured Cells Using a Rapid
and Sensitive LC-MS/MS Approach
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Abstract
Glycosaminoglycans
(GAGs), a family of polysaccharides widely distributed in eukaryotic
cells, are responsible for a wide array of biological functions. Quantitative
disaccharide compositional analysis is one of the primary ways to
characterize the GAG structure. This structural analysis is typically
time-consuming (1–2 weeks) and labor intensive, requiring GAG
recovery and multistep purification, prior to the enzymatic/chemical
digestion of GAGs, and finally their analysis. Moreover, 10<sup>5</sup>–10<sup>7</sup> cells are usually required for compositional
analysis. We report a sensitive, rapid, and quantitative analysis
of GAGs present in a small number of cells. Commonly studied cell
lines were selected based on phenotypic properties related to the
biological functions of GAGs. These cells were lysed using a commercial
surfactant reagent, sonicated, and digested with polysaccharide lyases.
The resulting disaccharides were recovered by centrifugal filtration,
labeled with 2-aminoacridone, and analyzed by liquid chromatography
(LC)-mass spectrometry (MS). Using a highly sensitive MS method, multiple
reaction monitoring (MRM), the limit of detection for each disaccharide
was reduced to 0.5–1.0 pg, as compared with 1.0–5.0
ng obtained using standard LC-MS analysis. Sample preparation time
was reduced to 1–2 days, and the cell number required was reduced
to 5000 cells for complete GAG characterization to as few
as 500 cells for the characterization of the major GAG disaccharide
components. Our survey of the glycosaminoglycanomes of the 20 selected
cell lines reveals major differences in their GAG amounts and compositions.
Structure–function relationships are explored using these data,
suggesting the utility of this method in cellular glycobiology