Glycosaminoglycanomics of Cultured Cells Using a Rapid and Sensitive LC-MS/MS Approach

*S Supporting Information 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, 105−107 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 chromato-graphy (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