Melamine foam assisted in-tip packed amine-functionalized titanium metal-organic framework for the selective...

Endogenous glycopeptides are significantly important in diverse pathological and physiological systems, but their direct analysis is severely hampered by their low abundance and presence of interfering species in biological fluids. In this study, we synthesized the amine-functionalized titanium metal-organic framework (NH₂-MIL-125(Ti)) by a simple hydrothermal method, characterized and used for glycopeptides enrichment. The designed separation media is highly hydrophilic and stable which is suitable for hydrophilic interaction chromatography (HILIC). To make the process smooth, simple, reliable, and robust, NH₂-MIL-125(Ti) crystals were packed in pipette-tip using hydrophilic melamine foam, as supporting frit. Free amine groups, present in the structure imparted hydrophilicity and a unique pattern of porosity, contributing to the size exclusion effect that excluded the large-sized proteins up to 1:700 peptide to protein ratio. The prepared MOF particles possessed regular porosity, high surface area, good hydrophilicity, and offered an in-tip flow-based set-up enhanced the enrichment performance for N-linked glycopeptides. The affinity material showed a detection limit of 1 fmol.µL^-1 and selectivity up to 1:1000 (HRP digest to BSA digest). Moreover, repeatability and reusability were evaluated up to five rounds of enrichment using the same affinity tip, and scanning electron microscopic images revealed no structural changes in the MOF crystals. Finally, the MOF packed in pipette tip was applied to selectively capture the N-linked endogenous glycopeptides from a healthy saliva sample and 64 unique endogenous glycopeptides were identified. These results demonstrated the excellent potential of NH₂-MIL-125(Ti) based affinity tip for glycopeptides which can be used to enrich trace glycopeptide biomarkers from the biological fluids.