Abstract
A novel glycoanalytical approach was developed in this study for the purification of fluorescently derivatized N-glycans. Polyethylene glycol (PEG) modified iron-nanoparticles were synthetized by the combination of sonochemical treatment and combustion method. The prepared nanomaterials were applied for a systematic clean-up optimization to maximize purification efficiency of 2-AA labelled glycans. PEG 1000 modified iron-oxalate was found to be the most effective for the selective enrichment of serum N-glycans providing high reproducibility. Different acetonitrile percentages for binding and washing steps were also tested to ensure the same relative peak areas compared to the unpurified sample. The generated novel clean-up strategy provides a potential route to use in-house synthetized magnetic nanoparticles for glycan sample preparation.
Highlights
The recognition of protein glycosylation importance in biomarker discovery research and biomanufacturing resulted in the need for novel glycoanalytical platforms [1]
Glycosylation of serum proteins is reportedly altered in many inflammatory [3] and malignant diseases [4] in ageing [5], pregnancy [6] and smoking [7] suggesting the need of high-throughput and simplified sample preparation platforms
Glycan profiling is most commonly performed by glycan release, fluorescent labelling, purification and analysis method which can be capillary electrophoresis or high-performance liquid chromatography (HPLC)
Summary
The recognition of protein glycosylation importance in biomarker discovery research and biomanufacturing resulted in the need for novel glycoanalytical platforms [1]. Glycan profiling is most commonly performed by glycan release, fluorescent labelling, purification and analysis method which can be capillary electrophoresis or high-performance liquid chromatography (HPLC). The derivatization step results in the stoichiometric attachment of a fluorescent tag to each glycan species enhancing detection sensitivity [8]. Several strategies have been developed in recent years for glycan purification such as solid phase extraction, precipitation, paper chromatography and gel filtration [10]. Most of these methods require sample preconcentration prior to analysis due to the high elution volume of the purification approaches. Ionic liquid modified hydrophilic MIONPs provided high detection sensitivity and enrichment recovery analyzing Hela exosome glycopeptides. Adalimumab and rituximab glycans were purified by different clean-up methods showing great comparability with conventional purification strategies
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