A novel ultra-sensitive method for the quantification of glycosaminoglycan disaccharides using an automated DNA sequencer.
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, the described method is a useful tool for the quantification of GAG disaccharides in low amounts, indicating advantages of obverse RP-HPLC and slab gel polyacrylamide electrophoresis in sensitivity, error-proneness, automation, and handling.
Vogel
Kay
K
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
Kuhn
Joachim
J
Kleesiek
Knut
K
Götting
Christian
C
D004187:Disaccharides / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D006025:Glycosaminoglycans / Q000032:analysis*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
D004187:Disaccharides; D006025:Glycosaminoglycans