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Glycobiology Advance Access originally published online on September 15, 2005
Glycobiology 2006 16(1):65-72; doi:10.1093/glycob/cwj037
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org

Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives

Daniel R. Studelska, Kari Giljum, Lynda M. McDowell and Lijuan Zhang1

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110

1 To whom correspondence should be addressed; e-mail: ljzhang{at}wustl.edu

Received on July 22, 2005; accepted on September 3, 2005

Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.

Key words: assay / glycosaminoglycan / hydrolysis / isolation / OPA


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L. M. McDowell, B. A. Frazier, D. R. Studelska, K. Giljum, J. Chen, J. Liu, K. Yu, D. M. Ornitz, and L. Zhang
Inhibition or Activation of Apert Syndrome FGFR2 (S252W) Signaling by Specific Glycosaminoglycans
J. Biol. Chem., March 17, 2006; 281(11): 6924 - 6930.
[Abstract] [Full Text] [PDF]


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