The disaccharide 2-sulfoglucuronate-N-acetyl-glucosamine (G2A0) was pre-labelled with the fluorescent dye 2-aminoacridone (AMAC).10 (link) AMAC-labelled G2A0 (12,5 nmol) was incubated with 100 µg protein of the appropriate homogenates in a final volume of 52,5 µl in 250 mM ammonium-acetate buffer pH 4.6 for 24 hours at 37°C. After centrifugation (15000 g, 4°C), the samples were analysed by C18-reversed-phase (RP)-chromatography in ammonium acetate buffer (60 mM, pH 5.6) with a flow rate of 1 mL/min with the Ettan LC system (GE Healthcare). The saccharides were eluted and fractionated with an acetonitrile gradient, in which AMAC-labelled disaccharides were detected by ultraviolet (UV) absorbance at 255 nm. The fractions containing AMAC-mediated fluorescence peaks were vacuum dried, resolved in 25 µl acetonitrile and analysed by nano-electrospray ionization mass spectrometry (ESI-MS) in a negative ion mode.
Protocol detail
Quantitative Analysis of Disaccharide Conjugates
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Acetonitrile
Ammonium acetate
Buffer
Centrifugation
Disaccharides
Dye 2
Electrospray ionization mass spectrometry
Fluorescence
Fluorescent dye
Glucosamine
Protein
Reversed phase chromatography
Saccharides
Vacuum
Corresponding Organization : Medical University of Graz
Other organizations : Manipal Academy of Higher Education, Kasturba Medical College, Manipal, University of Georgia, Bielefeld University, Emma Kinderziekenhuis, University of Amsterdam, Amsterdam University Medical Centers, Medical University of Vienna, Orthopaedic Hospital Speising
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Variable analysis
independent variables
- Incubation time (24 hours)
- Incubation temperature (37°C)
- Substrate conversion (AMAC-labelled disaccharides detected by UV absorbance at 255 nm)
- Molecular structure of AMAC-labelled disaccharides (analyzed by nano-electrospray ionization mass spectrometry in negative ion mode)
- Protein concentration (100 µg)
- Reaction buffer (250 mM ammonium-acetate buffer, pH 4.6)
- AMAC-labelled substrate concentration (12.5 nmol G2A0)
- Chromatography conditions (C18-reversed-phase chromatography, 60 mM ammonium acetate buffer, pH 5.6, 1 mL/min flow rate)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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