Screening for anticoagulant heparan sulfate octasaccharides and fine structure characterization using tandem mass spectrometry.

Heparan sulfate (HS) is a sulfated glycosaminoglycan located on the surface and extracellular matrix of mammalian cells. HS is constituted of highly N-sulfated domains (NS domains) interrupted by lower sulfation domains. The arrangement of these domains dictates the function of HS which is mainly involved in binding proteins and regulating their biological activities. Heparin, a heparan sulfate analogue present in mast cells, resembles the NS domains of HS but lacks the alternating high and low sulfation architecture. Because the NS domains that range up to hexadecasaccharide in size are the main protein binders, heparin has been used as a model for HS in protein binding studies. Heparan sulfate, however, is the more physiologically relevant modulator of growth factor-receptor interactions. In this work, liquid chromatography and mass spectrometry (LC-MS) were used to compare the compositions of affinity-purified heparin and HS octasaccharides with anticoagulant activities versus library octasaccharides. The fine structures of the biologically active HS compositions were then compared against those of library octasaccharides using low-energy collision-induced dissociation tandem mass spectrometry. This approach confirmed isomeric enrichment of these compositions and, most importantly, produces ions diagnostic of their biological activity.