This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparan sulfate (HS) is a linear polysulfated polysaccharide that is localized to the cell surface and extracellular matrix. This polysaccharide plays a significant biological role by modulating the activities of a wide array of proteins including coagulation enzymes, growth factors and morphogens. Several protein binding epitopes on HS have been characterized by traditional methods such as enzymatic digestion and disaccharide analysis. Here we describe a novel mass spectrometric based approach for characterization approach (HS) of protein binding epitopes. We have applied it to octasaccharides capable of binding Antithrombin III (ATIII). These represent the largest heparan sulfate oligosaccharides studied by on-line LC/MS2 to date. Porcine intestinal mucosa HS octasaccharide libraries were generated by exhaustive digestion with heparin lyase III followed by fractionation of the digestion material on preparative size exclusion chromatography (SEC) column. In order to purify the octasaccharides that bind ATIII, the octasaccharide (dp8) SEC fraction was mixed with ATIII and the unbound sugars were separated from the sugar-protein complex using SEC. Subsequently, the complex was trapped on a C-18 cartridge and the bound oligosaccharides eluted with 2M ammonium acetate. The characterization of the HS octasaccharide was done by LC/MS2 on a ThermoFisher Scientific LTQ-Orbitrap with on-line chromatographic separation prior to mass spectrometric detection using a capillary amide-80 column (TOSOH bioscience) packed in-house (250um X 15 cm) and an Advion Triversa Nanomate interface.