Precise structural analysis of O-linked oligosaccharides in human serum

摘要

O-glycans are suitable targets as novel and useful tumor markers. The structures of O-glycans in human sera from four healthy controls were precisely analyzed to obtain the reference O-glycan database. O-glycans were prepared from sera by hydrazine treatment followed by fluorescent labeling with aminopyridine and identified using twodimensional mapping, enzymatic digestion and mass spectrometry (MS) together with methanolysis and the use of newly synthesized sulfated oligosaccharides as standards. O-glycans, present at more than 0.01 of the total O-glycans, were analyzed, and 18 kinds of acidic and 2 kinds of neutral glycans were identified. NeuAca2-3Gal?1-3N-acetylgalactosamine (GalNAc) (61-64), NeuAca2-3Gal?1-3(NeuAca2-6) GalNAc (15-26) and Gal?1-3GalNAc (6-14) were major components while other sialylated glycans, Gal?1-3 (NeuAca2-6)GalNAc, Gal?1-4GlcNAc?1-6(NeuAca2- 3Gal?1-3) GalNAc and NeuAca2-3Gal?1-4GlcNAc?1-6(NeuAca2- 3Gal?1-3)GalNAc were relatively minor components, accounting for ～ 1-2. Very minor glycans accounting for 0.01-0.1 of the total include (i) the neutral glycan, Gal?1-4GlcNAc?1-6(Gal?1-3)GalNAc, (ii) sialylated glycans, having sialyl Tn antigen, agalacto and trisialylated structures, (iii) fucosylated glycans forming blood type H antigen, blood type A antigen, blood type B antigen, Lewis X antigen and sialyl Lewis X antigen and (iv) sulfated glycans, having 6-sulfo and 3'-sulfo structures. Two kinds of clinically applied tumor markers namely sialyl Tn antigen and sialyl Lewis X antigen in healthy controls sera were revealed to be present at ～ 0.1-0.2 of the total. However, other markers such as CA19-9 and DU-PAN-2 were not found, suggesting the relative amounts of these glycans to be 0.01. These detailed O-glycan profiles will help to find novel carbohydrate tumor markers.