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Heparan sulfate (HS) is a glycosaminoglycan expressed abundantly on cell surfaces and within extracellular matrixes of all adherent animal cells. HS consists of a repeating disaccharide unit of glucosamine and uronic acid residues with poly-disperse sulfation, N-acetylation and uronosyl epimerization. The elucidation of these patterns of modifications is crucial to understanding the functionality of HS but remains an exceptionally challenging task. HS from different tissues, i.e. aorta, lung, etc., was exhaustively digested with heparin lyase III, fractionated by size exclusion chromatography and analyzed by tandem MS using an LTQ-Orbitrap instrument. We applied automated on-line collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) of HS derived from different mammalian tissues.The goal of our work was to demonstrate that MS and MS/MS data contained sufficient information for (1) differentiation of isomeric glycoforms in the HS from different tissues, and (2) recognition of fragmentation patterns corresponding to HS structures. The MS/MS data were first treated with a classical interpretation approach. Under ideal conditions, complementary pairs of glycosidic bonds or cross-ring cleavages give definitive structural information. But due to the complexity of the data, presence of SO3 losses and presence of complex mixtures of isomeric glycoforms, only partial sequence information was achieved from the MS/MS data.Given the prevalence of glycoform isomers in mammalian tissue, we developed a novel approach based on bioinformatic tools and statistical algorithms. Using an HS oligosaccharide composition common to all tissues, MS/MS data were used as a means of differentiating glycoforms specific to different tissues. It was the feasible to identify the origin of HS based upon the MS/MS profile of each select glycoform. Enhanced glycoform and tissue specificity was achieved, allowing for significant improvement in HS characterization and classification compared with traditional methods.