Analysis of 3- O -Sulfated Heparan Sulfate Using Isotopically Labeled Oligosaccharide Calibrants.

The 3- O -sulfated glucosamine in heparan sulfate (HS) is a low-abundance structural component, but it is a key saccharide unit for the biological activities of HS. A method to determine the level of 3- O -sulfated HS is lacking. Here, we describe a LC-MS/MS based method to analyze the structural motifs. We determined the levels of 3- O -sulfated structural motifs from pharmaceutical heparin manufactured from bovine, porcine, and ovine. We discovered that saccharide chains carrying 3- O -sulfation from enoxaparin, an FDA-approved low-molecular weight heparin, displayed a slower clearance rate than non-3- O -sulfated sugar chains in a mouse model. Lastly, we detected the 3- O -sulfated HS from human brain. Furthermore, we found that a specific 3- O -sulfated structural motif, tetra-1, is elevated in the brain HS from Alzheimer's disease patients ( n = 5, p = 0.0020). Our method offers a practical solution to measure 3- O -sulfated HS from biological sources with the sensitivity and quantitative capability.