A novel ultra-sensitive method for the quantification of glycosaminoglycan disaccharides using an automated DNA sequencer

Electrophoresis
Kay VogelChristian Götting

Abstract

Analysis of glycosaminoglycans (GAGs) is of increasing importance concerning alterations in extracellular matrix composition and selectivity of glomerular basement membrane. In this report we describe the analysis of chondroitin sulfate disaccharides as an example of GAG delta disaccharide analysis using standard DNA sequencing equipment (DNA sequencer-assisted GAG disaccharide separation, DSA-GAGS). The presented methodology allows nanomolar quantification of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-derived GAG disaccharides. In comparison to RP-HPLC the established method is much more sensitive, showing detection limits of 38 fmol/microL. Variation coefficients were approximately 10%, enabling exact quantifications after run times of 17 min at 30 degrees C and an electrophoresis voltage of 15 kV; using a capillary DNA sequencer, available in many molecular laboratories, presented advantages like automated sample injection, opportunity of high-throughput analyses, separation of even sulfated disaccharide epimers, and the possibility of using APTS-derived fucose as an internal standard. Furthermore, highly reproducible retention times rendered easy identification of specific signals (SD 0.02). With regard to these results, t...Continue Reading

References

Jun 26, 1999·The Journal of Investigative Dermatology·C GöttingK Kleesiek
Jan 30, 2002·Clinica Chimica Acta; International Journal of Clinical Chemistry·Christian GöttingKnut Kleesiek
Dec 7, 2002·Molecular Human Reproduction·Christian GöttingKnut Kleesiek
Mar 17, 2004·Journal of Pharmaceutical and Biomedical Analysis·Evgenia G KarousouAlberto Passi
Apr 20, 2004·Cellular and Molecular Life Sciences : CMLS·I B H Wilson

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Citations

Jun 19, 2008·Analytical and Bioanalytical Chemistry·Angelo ZinelluCiriaco Carru
Apr 5, 2007·Nature Protocols·Wouter LaroyNico Callewaert
May 22, 2008·Molecular BioSystems·Emiliano GemmaAlison N Hulme
Feb 5, 2010·Biological Chemistry·Dieter VanderschaegheNico Callewaert
Apr 5, 2011·Annual Review of Analytical Chemistry·John F Rakus, Lara K Mahal
Nov 28, 2008·Electrophoresis·Alicia M HitchcockJoseph Zaia

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