期刊
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
卷 837, 期 1-2, 页码 76-86出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2006.04.013
关键词
bio-gel; heparan sulfate proteoglycans; heparin-binding proteins; heparinase; heparin oligosaccharides; mass spectrometry; size-exclusion chromatography; superdex; van Deemter plot
资金
- NCRR NIH HHS [P41RR10888] Funding Source: Medline
- NHLBI NIH HHS [R01HL74197] Funding Source: Medline
Recent findings on specific and non-specific interactions of glycosaminoglycans (GAGs) accentuate their pivotal role in biology and the call for improved sequencing tools. The present study evaluates size-exclusion chromatography (SEC) of heparin oligosaccharides at high and low pressure, requiring amounts as low as 0.2 microgram, using conventional UV detection after depolymerization with heparin lyases. Because of their high charge at physiological pH, SEC elution volumes of heparin oligosaccharides depend on both molecular size and charge repulsion from the matrix. As a consequence, SEC elution volumes of GAGs are smaller than those of globular proteins of similar molecular weight, and this might be exploited. Accordingly, larger heparin oligosaccharides are best separated according to their size at high ionic strength of the mobile phase (> 30 mM); in contrast, disaccharides are best separated according to their charge at low ionic strength, compatible with on-line coupling to mass spectrometry. Optimized SEC affords separation of characteristic heparin trisaccharides that contain uronic acid at the reducing end and suggest cellular storage of heparin as a free glycan. (c) 2006 Elsevier B.V. All rights reserved.
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