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In particular, κ-casein glycomacropeptide and lactophorin, that have several O-glycans, are known to exist in whey necessary protein. Here, we report an isolation approach to O-glycopeptides bearing disialyl core 1 kind and core 2 kind glycan moieties from commercially offered whey necessary protein products using proteose peptone removal, enzymatic food digestion (with trypsin or thermolysin), and sequential high-performance liquid chromatography purification. We were able to isolate a f