Quantitative N-glycomics can reveal abnormal expression of N-glycan in diseases. However, mass spectrometry (MS)-based N-glycome quantitative analysis is still technically challenging. To achieve the quantitation of N-glycome with high accuracy and sensitivity, it is required to efficiently label the N-glycans with isotopic tags and selectively enrich N-glycans to avoid suppression from other substances. Herein, we developed an integrated pipeline that combines isotopically fluorous tag labeling and fluorous solid-phase extraction to quantitatively analyze the N-glycome by MS. In this strategy, the N-glycans were labeled with light and heavy aminoxy-functionalized fluorous tags (PFBHA and PFBHA-d(2)) through the oxime click reaction. Then through the fluorous solid-phase extraction, the fluorous tag labeled N-glycan could be purified from contaminants like salts and proteins for the following quantitative analysis by mass spectrometry. This new approach enables selective purification (molar ratio of glycan to protein at 1:100) and accurate (R-2 > 0.99) and reproducible (coefficient of variation (CV)) < 25%, n = 6) quantitation of N-glycans within 2 orders of magnitude. Uniquely, diagnostic ions (D and [D-221]) were generated in tandem MS analysis after fluorous tags labeling, which could be used to deduce the composition of the 6-antenna and to distinguish isomers. Finally, this strategy was successfully applied to analyze the N-glycan changes in human serum associated with hepatocellular carcinoma (HCC). Fifteen N-glycan compositions with bisecting GlcNAc, sialic acid, and core fucosylation showed significant differences in HCC serum.
[1]Fudan Univ, Shanghai Canc Ctr, Minist Publ Hlth, Shanghai 200032, Peoples R China;
[2]Fudan Univ, Dept Chem, Minist Publ Hlth, Shanghai 200032, Peoples R China;
[3]Fudan Univ, Inst Biomed Sci, Minist Publ Hlth, Shanghai 200032, Peoples R China;
[4]Fudan Univ, Key Lab Glycoconjugates Res, Minist Publ Hlth, Shanghai 200032, Peoples R China
(8.0+极速模式)