Printed covalent glycan array for ligand profiling of diverse glycan binding proteins

Blixt et al. 10.1073/pnas.0407902101.

Supporting Information

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Fig. 7. Complete listing of printed glycans (1-200).

Supporting Methods

Pronase Digestion of Bovine Pancreatic Ribonuclease B

Bovine pancreatic ribonuclease B (540 mg, Sigma lot 060K7650) was dissolved in 5 ml of 0.1 M Tris + 1 mM MgCl₂ + 1 mM CaCl₂, pH 8.0

Pronase (108 mg, Calbiochem lot B 50874) was added to give a ratio by weight of five parts glycoprotein to one part pronase. It was incubated at 60°c for 3 h.

A second dose of 108 mg of pronase was added and incubated at 37°c for another 3 h after which it was boiled for 30 min, cooled, and centrifuged.

The sample was loaded onto 20 ml of freshly prepared Con A in 0.1 M Tris, 1 mM MgCl_2, and 1 mM CaCl_2, pH 8.0, washed, and eluted with 200 ml of 0.1 M methyl-a-D-mannopyranoside (Calbiochem lot B37526)

The Con A-eluted sample was purified on a Carbograph solid-phase extraction column (Alltech Associates, 1,000 mg, 15 ml) and eluted with 30% acetonitrile + 0.06% trifluoroacetic acid. It was dried and reconstituted in 1 ml of water. Mass analysis was done by MALDI, and glycan quantitation was done by phenol sulfuric acid assay.

Separation of Fractions on a Dionex BioLC

Twenty microliters of the pronase-digested ribonuclease B was injected on a Dionex BioLC by using a PA-100 column and eluted with the following gradient: solution A, 0.1 M NaOH, solution B, 0.5 M NaOAc in 0.1 M NaOH; 0% B for 3 min, then a linear gradient from 0% B to 6.7% B in 34min. The individual peak fractions were collected and purified on Carbograph solid-phase columns (Alltech Associates, 150 mg, 4 ml) by eluting with 80% acetonitrile containing 0.1% trifluoroacetic acid. They were dried and reconstituted in water. Final mass analysis and glycan quantitation were performed.

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