Chemical degradation ~Partial acetolysis

 Peracetylate a pyridylamino (PA-) sugar chain (0.1–10 nmol) with a mixture of 20 μL of acetic anhydride and 20 μL of pyridine at 100°C for 15 min.

 Evaporate the excess reagent.

 Add 20 μL of a mixture of acetic anhydride, acetic acid, and sulfuric acid (10:10:1).

 Incubate at 37°C for 12 h.

 Neutralize with 4 μL of pyridine.

 Evaporate the excess reagent.

 Add 0.4 mL of a 50% saturated sodium bicarbonate solution.

 Extract three times with each 0.5 mL of chloroform.

 Combine extracted chloroform solutions, and dry with anhydrous sodium sulfate.

 Divide into two aliquots. (One is for analysis of reducing end fragment, and the other is for analysis of non-reducing end fragments.)

 Remove the chloroform under reduced pressure.

 Hydrazinolyze with 0.1 mL of anhydrous hydrazine at 100°C for 22 h.

 Remove the excess hydrazine by repeated evaporation with toluene.

 Add 8 μL of acetic anhydride and 200 μL of a saturated sodium bicarbonate solution.

 Incubate on ice for 30 min.

 Add 0.5 g of Dowex 50x2 (H⁺).

 Wash the resin with 5 mL of DDW.

 Elute the PA-sugar chain with 3 mL of 1.5 M aqueous ammomia.

 Concentrate the eluate to dryness.

 Analyze the PA-sugar chain derived from the reducing end by HPLC as described in the other protocol (Fluorescent labeling of glycans and HPLC analysis).

 Remove the chloroform under reduced pressure.

 Dissolve in 50 μL of methanol.

 Add 20 μL of 0.2% sodium methoxide-methanol solution.

 Incubate at room temperature for 30 min.

 Acidify with 1% acetic acid, and dry up.

 Pyridylaminated with 10 μL of coupling solution and 35 μL of reducing reagent as described in the other protocol (Fluorescent labeling of glycans and HPLC analysis).

 Add Dowex 50x2 (H⁺) to bring the pH to 3.

 Wash the resin with 10 bed volumes of DDW.

 Elute the PA-sugar chain with 6 bed volumes of 1.5 M aqueous ammomia.

 Dry up.

 Co-evaporate repeatedly with 50% triethylamine solution under reduced pressure.

 Purify the PA-sugar chain by paper electrophoresis (Whatman No.1) at 900 V/25cm for 50 min in a mixture of acetic acid, pyridine and water (2:3:60, pH 5.0).

 Analyze the PA-sugar chain derived from the non- reducing end by HPLC as described in the other protocol (Fluorescent labeling of glycans and HPLC analysis).