Dual isotopic labeling combined with fluorous solid-phase extraction for simultaneous discovery of neutral/sialylated N-glycans as biomarkers for gastric cancer.

Mass spectrometry analysis coupled with stable isotope labeling is a powerful strategy for comparative analysis of N-glycans for glycan biomarker discovery. Here, a novel method combining dual isotopic labeling and fluorous solid-phase extraction was developed, enabling selective enrichment, simultaneous quantitation and recognition of neutral/sialylated glycans from serum samples by mass spectrometry. The isotopic label of fluorous compound on the reducing end of glycans acts as an enrichment tag and provides mass difference between neutral glycans from different samples, while the isotopic label on the non-reducing end of glycans protects the sialic acid residue and provides additional mass difference for sialylated glycans. Therefore, the neutral/sialylated glycans could be simultaneously enriched through the fluorous solid-phase extraction (FSPE) and quantified by mass spectrometry. This method provided a good linearity (R > 0.99) and high reproducibility (CV < 20%) within 2 orders of magnitude in the dynamic range. Finally, this strategy was successfully applied to investigate the N-glycome alteration in serum associated with gastric cancer (GC). Bisecting GlcNAc and triantennary glycan compositions were found to be significantly changed between GC cases (n = 50) and healthy control, indicating the great potential to be novel biomarkers for GC early diagnosis.