Untangling Heparan Sulfate 3-<i>O</i>-Sulfation Using a Novel Offline Cationic-Peptide Affinity Enrichment, Followed by HILIC-cIM-MS

Heparan sulfate (HS) is a linear polysaccharide that modifies proteoglycans. HS biosynthesis is regulated in a spatiotemporal manner, leading to structural diversity, including variable de-<i>N</i>-acetylation, <i>N</i>-sulfation, hexuronic acid C5 epimerization, and 2-<i>O</i>-, 6-<i>O</i>-, and 3-<i>O</i>-sulfation. Specific structural motifs within HS chains offer multiple specific binding sites for protein partners. The occurrence of HS 3-<i>O</i>-sulfation is relatively rare; however, there is accumulating evidence identifying the importance of this low-abundance modification in many different biological scenarios. Initially described as a key determinant for binding and activation of antithrombin, and more recently, as a coreceptor for viral infection, 3-<i>O</i>-sulfation has been associated with the progression of several neurological disorders. The analytical ability to study the biological roles of HS 3-<i>O</i>-sulfation is hindered by its low abundance within HS chains and the complex isomeric nature of highly sulfated HS, which places a burden on the tandem mass spectrometry step for assigning saccharide structures. In this context, we developed a specific cationic peptide-affinity method for 3-<i>O</i>-sulfation enrichment, followed by hydrophilic interaction liquid chromatography-cyclic ion mobility mass spectrometry analysis (HILIC-cIM-MS). We first demonstrated the high specificity of this approach to capture 3-<i>O</i>-sulfated HS oligosaccharides within complex mixtures. We next showed the influence of specific sulfate and epimerization patterns on HS binding selectivity. Finally, we used the enrichment strategy to analyze 3-<i>O</i>-sulfated HS oligosaccharides from heparin lyase III-digested HS from porcine intestinal mucosa (HSPIM). We concluded that this enrichment method was useful to guide new studies to reveal the biological roles of 3-<i>O</i>-sulfation and to elucidate new HS structural motifs.