A Compact Method Specialized for Full Analysis of the Glycan Composition of Jellyfish Mucin Using Limited Degradation Followed by High-Resolution Mass Spectrometry

A compact and specialized method for analyzing the O-glycan composition of jellyfish mucin (Q-mucin) has been newly developed. Q-mucin was analyzed using high-resolution mass spectrometry (HRMS) with electrospray ionization (ESI), combined with limited degradation (LD) by endoprotease Glu-C. Direct analysis of the solution after enzymolysis caused unwanted dissociation of glycan adducts from tandem repeats (TRs). However, the introduction of HPLC prior to ESI, utilizing a reverse-phase column with an aqueous solution of formic acid and acetonitrile as the mobile phase, successfully minimized the source dissociation of many glycan adducts from TRs. As a result, we established a rapid and cost-effective protocol for analyzing the O-glycans in Q-mucin over a wide dynamic range. Q-mucin contains two types of TRs that are commonly shared by a large number of jellyfish species. However, the residual challenge lies in understanding the O-glycan composition, which varies depending on species and ecological factors. For systematic studies across a vast number of species, the newly developed protocol, which does not rely on complex analytical algorithms, proves highly suitable. Given its superior time efficiency and accuracy compared to large-scale mass analysis programs designed for general use, we propose that the development of purpose-oriented methods holds significant value for specialized studies using mass spectrometry.