Highly Efficient Identification of O‐GalNAc Glycosylation by an Acid‐Assisted Glycoform Simplification Approach |
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Authors: | Xin You Hongqiang Qin Jiawei Mao Yu Tian Mingming Dong Zhimou Guo Xinmiao Liang Liming Wang Yan Jin Mingliang Ye |
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Institution: | 1. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, 116023 Dalian, China;2. University of Chinese Academy of Sciences, 100049 Beijing, China;3. Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, The Second Hospital of Dalian Medical University, Dalian Medical University, 116023 Dalian, Liaoning, China |
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Abstract: | Compared with N‐linked glycosylation, the analysis of O‐GalNAc glycosylation is extremely challenging due to the high structure diversity of glycans and lack of glycosidases to release O‐GalNAc glycans. In this work, a glycoform simplification strategy by combining HILIC enrichment with chemical de‐sialylation to characterize O‐GalNAc glycosylation of human serum is presented. This method is first validated by using the bovine fetuin as the test sample. It is found that more than 90% of the sialic acid residues can be removed from bovine fetuin by the acid‐assisted de‐sialylation method, which significantly simplifies the glycan structure and improves identification sensitivity. Indeed, the number of identified peptide backbones increases nearly one fold when this strategy is used. This method is further applied to analyze the human serum sample, where 185 O‐GalNAc modified peptide sequences corresponding to 94 proteins with high confidence (FDR (false detection rate) <1%) are identified. This straight forward strategy can significantly reduce the variations of glycan structures, and is applicable to analysis of other biological samples with high complexity. |
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Keywords: | O‐GalNAc HILIC enrichment N‐acetyl‐neuraminic acid |
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