Analysis of Heparan Sulfate Oligosaccharides with Ion Pair-Reverse Phase Capillary High Performance Liquid Chromatography-Microelectrospray Ionization Time-of-Flight Mass Spectrometry
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- 作者：Balagurunathan Kuberan ; Miroslaw Lech ; Lijuan Zhang ; Zhengliang L. Wu ; David L. Beeler ; and Robert D. Rosenberg
- 刊名：Journal of the American Chemical Society
- 出版年：2002
- 出版时间：July 24, 2002
- 年：2002
- 卷：124
- 期：29
- 页码：8707 - 8718
- 全文大小：178K
- 年卷期：v.124,no.29(July 24, 2002)
- ISSN：1520-5126
文摘
Heparan sulfate, a cell surface bound glycosaminoglycan polysaccharide, has been implicatedin numerous biological functions. Heparan sulfate molecules are highly complex and diverse, yet deceivinglylook simple and similar, rendering structure-function correlation tedious. Current chromatographic andmass spectrometric techniques have limitations for analyzing glycosaminoglycan samples that are in lowabundance and that are large in size, due to their highly acidic nature arising from a large number ofsulfate and of carboxylate groups. A new methodology was developed using capillary ion-paired reverse-phase C18 HPLC directly coupled to ESI-TOF-MS to address the above issues. On the basis of HSdisaccharide analysis, dibutylamine was found to be the best suited for HS analysis among many ion-pairing agents investigated. Next, analysis of oligosaccharides derived from heparosan, the precursor forheparan sulfate, was undertaken to demonstrate its greater applicability in a more complex structuralanalysis. The established chromatographic conditions enabled the characterization of heparosan oligosaccharides of sizes up to tetracontasaccharide with high resolution in a single run and were amenable tonegative ion electrospray MS in which sodium adduction and fragmentation were avoided. To date, theseare the largest nonsulfated HS precursor oligosaccharides to be characterized by LC/MS. Finally, the currentmethodology was applied to the characterization of the biologically important ATIII binding pentasaccharideand its precursors, which differ from each other by sulfation pattern and/or degree of sulfation. All of thesepentasaccharides were well-resolved and characterized by the LC/MS system with 34SO4 as a mass spectralprobe. This newly developed methodology facilitates the purification and rapid characterization of biologicallysignificant HS oligosaccharides, and will thus expedite their synthesis. These findings should undoubtedlypave the way in deciphering multiple functional arrangements, ascribed to many biological activities, whichare predictably embedded in a single large chaotic, yet well-organized HS polysaccharide chain.Development of newer techniques for HS oligosaccharide analysis is greatly needed in the postgenomeera as attention shifts to the functional implications of proteins and carbohydrates in general and HS inparticular.