Improved Hydrophilic Interaction Chromatography LC/MS of Heparinoids Using a Chip with Postcolumn Makeup Flow

摘要

Heparan sulfate (HS) and heparin are linear, heteroge-nneous carbohydrates of the glycosaminoglycan (GAG)nfamily that are modified by N-acetylation, N-sulfation,nO-sulfation, and uronic acid epimerization. HS interactsnwith growth factors in the extracellular matrix, therebynmodulating signaling pathways that govern cell growth,ndevelopment, differentiation, proliferation, and adhesion.nHigh-performance liquid chromatography (HPLC)-chip-nbased hydrophilic interaction liquid chromatographymass spectrometry has emerged as a method for analyzingnthe domain structure of GAGs. However, analysis of highlynsulfated GAG structures decasaccharide or larger in sizenhas been limited by spray instability in the negative-ionnmode. This report demonstrates that addition of postcol-numn makeup flow to the amide-HPLC-chip configurationnpermits robust and reproducible analysis of extendednGAG domains (up to degree of polymerization 18) fromnHS and heparin. This platform provides quantitativeninformation regarding the oligosaccharide profile, degreenof sulfation, and nonreducing chain termini. It is expectednthat this technology will enable quantitative, comparativenglycomics profiling of extended GAG oligosaccharidendomains of functional interest.