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 GAGs are expressed on the surfaces of all adherent animal cells and are known to bind many growth factors and growth factor receptors. As part of an ongoing effort to develop methods to enable the characterization of the heparan sulfate structural phenotype on cell surfaces, we are using heparin lyases to digest heparan sulfates and are studying the material which has been observed to resist digestion. Isolated and purified HS chains were subjected to depolymerization using combinations of heparin lyases I, II, and III. The extent of each reaction was monitored using UV spectrophotometry to select appropriate time points for MS analysis. Oligosaccharides were purified by ion-pairing reversed phase chromatography. I this technique, the negatively charged oligosaccharides are paired with positively charged amines, thereby increasing their hydrophobicity to the point that they may be separated using a reversed phase column. The eluted peptides were cleaned using a cation exchange resin to allow their analysis using negative ion nano ESI MS. Heparin lyases cleave by an eliminative mechanism to produce a 4,5-unsaturated acid residue at the new non-reducing oligosaccharide end, generating a unique absorbance at 232 nm that provides a means of following the extent of digestion using spectrophotometry. Using SEC-LC-MS, this progression can be monitored from initiation to completion, providing specific structural information on the lyase products. Using the mass spectrometer as the detector, molecular detail of intermediate enzyme products can be elucidated, providing structural information of HS as it is depolymerized. This is significant in determining enzymatic conditions sufficient to produce oligomers of sufficient size such that they could contain protein binding regions. Post-column splitting of the SEC effluent allows fractions containing these regions to be isolated for subsequent affinity studies and tandem mass spectrometric analysis. As a result, digestion products are simultaneously analyzed and purified for further tandem analysis.