Deciphering rare 3-O-sulfated motifs in heparan sulfate (HS) is the key to understanding critical biological processes, such as viral infection and tumor progression. However, the structural analysis of these elusive structures remains highly challenging due to their low abundance and the limitations of conventional analytical approaches. In this study, we report the application of human lysosomal arylsulfatase G (ARSG), a sulfatase specifically targeting 3-O-sulfated glucosamine residues, as a novel enzymatic tool, which, in combination with heparinase and heparanase digestion, facilitates the identification of 3-O-sulfated glucosamine moieties within HS. Coupling ARSG-mediated regioselective desulfation with hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) enables the efficient detection and quantification of 3-O-sulfated oligosaccharides in HS and heparin derivatives. This approach overcomes a major analytical barrier in HS characterization and provides new insights into the distribution and functional roles of 3-O-sulfation. The integration of ARSG expands the enzymatic toolkit available for HS structural characterization and represents a significant advance toward elucidating the structure-function relationships of HS in health and disease.
Poyer et al. (2026) studied this question.