High sensitivity profiling of N-glycans from mouse serum using fluorescent imidazolium tags by HILIC electrospray ionisation spectrometry
N -linked glycosylation is a ubiquitous protein post-translational modification in which aberrant glycan biosynthesis has been linked to severe conditions like cancer. Accurate qualitative and quantitative analysis of N -glycans are crucial for investigating their physiological functions. Owing to the intrinsic absence of chromophores and high polarity of the glycans, current detection methods are restricted to liquid chromatography and mass spectrometry. Herein, we describe three new imidazolium-based glycan tags: 2’GITag, 3’GITag, and 4’GITag, that significantly improve both the limit of detection and limit of quantification of derivatized oligosaccharides, in terms of fluorescence intensity and ionisation efficiency. Our top-performing derivatisation agent, 4’GITag, shifted the detection sensitivity range from high femtomole to sub-femtomole levels in ESI-MS compared to traditional glycan label, 2AB, enabling the identification of 24 N -glycans in mouse serum, including those bearing sialic acids. Additionally, 4’GITag stabilized Na-salt forms of sialic acids, simplifying the simultaneous analysis of neutral and negative charged N -glycans significantly, avoiding the need for complex derivatisation procedures typically required for the detection of sialylated species. Overall, the favorable performance of imidazolium tags in the derivatisation and sensitive profiling of glycans has the potential for labeling tissue or live cells to explore disease biomarkers and for developing new targeted therapeutic strategies.
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