The release of sulfates by the Sulfs with respect to the total sulfate R428 content of HS has been estimated as 5-7% from in vitro experiments using QSulf1. These changes, though relatively small in magnitude, have been associated with alterations in the binding of HS to extracellular signaling molecules. In the cases of GDNF, BMP, Shh, and Wnt, Sulfs serve as positive regulators and enhance signaling. The opposite is true for FGF2, HBEGF, HGF, and TGF-b. The long list of important growth factors and morphogens, the activities of which are modulated by the Sulfs, indicates the important roles of these extracellular HS editors in proliferation, migration, and differentiation. Depending on the context, Sulfs can serve as oncogenic effectors or tumor suppressors, and reports detailing the role of Sulfs in cancer are numerous. Most recently, Sulf2 was identified as a transcriptional target of the tumor suppressor p53. In light of findings that changes in Sulf expression levels influence HS biosynthesis, the aim of the current study was to extend knowledge of Sulf modification of HS by analyzing various HS substrates that have been modified by recombinant human Sulf2. These studies aimed to understand how Sulfs edit HS isolated from different a certain tissue contexts, and the use of a purified enzyme preparation eliminated the variables, such as altered expression of HS biosynthetic enzymes, that confounded the study of Sulf activity in Sulf over-expressing or knockout animals or cell lines. The current experiments also provided the first assessment of HSulf2 activity at HS chain termini, regions that have been implicated in biochemically important protein-binding events that control developmental processes. The abundances of these structures were considerably higher in the bovine samples, and were too low in the murine samples for accurate quantitation. This is likely explained by the amount of HS injected onto the LC/MS system and the intensity of the tetrasaccharides was proportional to the overall signal for all disaccharides observed in the LC/MS datasets. With the exception of bovine intestine, an increase in the abundance of and was observed for all samples after treatment with HSulf2, as shown in Fig. 8. The abundances of and are observedtoincreaseinthebovineHSpopulationstreatedwithHSulf2, except for bovine intestine HS. Interestingly, when compared to its internal counterpart, displayed a greater fold-change increase in abundance. These lyase-resistant oligosaccharides represent a small percentage of domains in HS chains. It is known that disaccharide repeats containing a 3O-sulfated GlcN residue resist heparin lyase cleavage. Such residues are required for anticoagulant function of heparin/HS. The changes in the abundances observed in Fig. 8 that result from HSulf2 digestion indicated that the presence of 6O-sulfate groups influences the susceptibility of the oligosaccharides to lyase digestion.