Urine samples were defrosted at 4 °C and mixed well using a vortex mixer. A 400-μL aliquot of each sample was desalted by passing through a 3 kDa molecule weight cutoff (MWCO) spin column and washed twice with distilled water. A series of urine samples were also prepared for method validation by adding aqueous solutions containing varying amounts of standard CS and HS affording final concentrations of 50–500 ng/mL.
The casing tubes were replaced before 150 μL of digestion buffer (50 mM ammonium acetate containing 2 mM calcium chloride adjusted to pH 7.0) was added to the filter unit. Recombinant heparin lyase I, II, III (pH optima 7.0–7.5) and recombinant chondroitin lyase ABC (10 mU each, pH optimum 7.4) were added to each sample and mixed well. The samples were all placed in a water bath at 37 °C for 2 h, after which enzymatic digestion was terminated by removing the enzymes by centrifugation. Under these reaction conditions, these lyases could completely depolymerize their GAG substrates (in amounts of over 100 μg) into products containing each class of GAG disaccharides. The filter unit was washed twice with 100 μL distilled water and the filtrates, containing the disaccharide products, were dried via vacuum centrifuge and stored at –20 °C.
The dried samples were AMAC-labeled by adding 10 μL of 0.1 M AMAC in DMSO/acetic acid (17/3,V/V) incubating at room temperature for 10 min, followed by adding 10 μL of 1 M aqueous NaBH3CN and incubating for 1 h at 45 °C. A mixture containing all 17-disaccharide standards prepared at 1250 ng/mL was similarly AMAC-labeled and used for each run as an external standard. After the AMAC-labeling reaction, the samples were centrifuged and each supernatant was recovered and an equal volume of DMSO:acetic acid:distilled water (17:3:20) was added to each. Samples were stored in a light-resistant container at room temperature until analyzed via LC-MS/MS.
The casing tubes were replaced before 150 μL of digestion buffer (50 mM ammonium acetate containing 2 mM calcium chloride adjusted to pH 7.0) was added to the filter unit. Recombinant heparin lyase I, II, III (pH optima 7.0–7.5) and recombinant chondroitin lyase ABC (10 mU each, pH optimum 7.4) were added to each sample and mixed well. The samples were all placed in a water bath at 37 °C for 2 h, after which enzymatic digestion was terminated by removing the enzymes by centrifugation. Under these reaction conditions, these lyases could completely depolymerize their GAG substrates (in amounts of over 100 μg) into products containing each class of GAG disaccharides. The filter unit was washed twice with 100 μL distilled water and the filtrates, containing the disaccharide products, were dried via vacuum centrifuge and stored at –20 °C.
The dried samples were AMAC-labeled by adding 10 μL of 0.1 M AMAC in DMSO/acetic acid (17/3,V/V) incubating at room temperature for 10 min, followed by adding 10 μL of 1 M aqueous NaBH3CN and incubating for 1 h at 45 °C. A mixture containing all 17-disaccharide standards prepared at 1250 ng/mL was similarly AMAC-labeled and used for each run as an external standard. After the AMAC-labeling reaction, the samples were centrifuged and each supernatant was recovered and an equal volume of DMSO:acetic acid:distilled water (17:3:20) was added to each. Samples were stored in a light-resistant container at room temperature until analyzed via LC-MS/MS.
