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. Heparin and heparan sulfate (HS) are glycosaminoglycans (GAGs) comprised of alternating glucuronic acid and N-acetylglucosamine units. These molecules are most widely recognized for their ability to bind proteins and modulate cellular events such as differentiation, blood coagulation, pathogenesis, and proliferation. Full understanding of these events requires knowledge of GAG structure. Although chip-based hydrophilic interaction chromatography (HILIC) is a powerful means of separating GAGs for on-line LC/MS, the negative-ion spray becomes unstable in high percent aqueous mobile phases. This work demonstrates the effectiveness of a chip designed with post-column addition of makeup LC flow to enable stable electrospray throughout the HILIC gradient. The result is a system with substantially increased spray stability and range of analytes that may be effectively analyzed. Porcine intestinal mucosa derived heparin was partially digested (30% reaction completion) with heparin lyase I. The resultant oligosaccharides were separated on a 1.75-meter size exclusion chromatography (SEC) column and pooled based on degree of polymerization. Bovine kidney HS was partially digested (10 or 20% reaction completion) to oligosaccharides with a mixture of heparin lyases I, II, and III. HS oligosaccharides as well as the low molecular weight heparin drugs Fragmin and Lovenox were purified by SEC-HPLC using a Superdex Peptide column. Samples were injected onto a custom-packed amide make-up flow HPLC-chip that was interfaced with an Agilent 6520 QTOF. Glycan compositions were assigned using a Microsoft Excel database and abundances calculated based on integrated peak area in the MS mode.
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