Selective enrichment and profiling of GlcNAc-containing oligosaccharides in Ophiocordyceps sinensis (Berk.) across five developmental stages using WGA-functionalized NH2-MIL-125

Glycosaminoglycans (GAGs)-like oligosaccharides in Ophiocordyceps sinensis ( O. sinensis ) are potential mediators of fungus-host interactions. However, their characterization is severely hindered by co-existing neutral saccharides and the low abundance N-acetylglucosamine (GlcNAc)-containing species (0.67%) of total monosaccharide. To overcome this matrix interference, we engineered a dual-interaction affinity platform (NH 2 -MIL-125-WGA) by covalently immobilizing Wheat Germ Agglutinin (WGA) onto an amino-functionalized titanium metal-organic framework. The optimized material achieved a high WGA loading capacity of 413.14 ± 16.93 μg/mg and maintained over 80% recovery of target acidic glycans, even facing a 1000-fold excess of neutral sugars. The elimination of matrix suppression enabled the identification of 52 previously undetectable GAG-like oligosaccharides in O. sinensis via tandem mass spectrometry. Furthermore, comparative glycomic profiling across five developmental stages (mycelium, zombie larva, fruiting bodies, and spore spread period) revealed that highly sulfated and polymerized oligosaccharides were significantly upregulated during the infective mycelium stage but were notably downregulated during the transition to the reproductive fruiting body stage. These findings demonstrate the capability of the NH 2 -MIL-125-WGA platform for trace glycan analysis and suggest that dynamic variations in GAG content and sulfation patterns are critical to the fungal life cycle.