The reaction mixture containing 200 |M CMP-NANA, 200 |M inhibitor (calculated as the click reaction was completed), 1 mM Gal-(GlcNAc)3-aminooxy (ao) tryptophanyl arginine methyl ester (WR), 12 mM sodium cacodylate buffer (pH 7.4), 0.15 mg/ml bovine serum albumin (BSA), 0.03% Triton CF-54, and 0.5 mU sialyltransferase, was adjusted to a total volume of 25 |l. The reaction mixture was incubated at 37 °C for 30 min. After incubation, 100 |l of CH3CN was added to stop the reaction, and 125 |l of water was added to the mixture to have a total volume of 250 |l. To the 20 |l of 2.5-dihydroxy benzoic acid DHB solution (10 mg/ml DHB in 30% CH3CN) was added 2 |l each of reaction mixture and deuterium-labeled internal standard, and then 0.5 |l of mixed solution was mounted on the MALDI MS target plate.
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