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Click-iG: Simultaneous Enrichment and Profiling of Intact N-linked,O-GalNAc,and O-GlcNAcylated Glycopeptides |
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| Authors: | Dr Jialin Liu Dr Bo Cheng Dr Xinqi Fan Xinyue Zhou Jiankun Wang Dr Wen Zhou Hengyu Li Dr Wenfeng Zeng Prof Pengyuan Yang Prof Xing Chen |
| Institution: | 1. College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking-Tsinghua Center for Life Sciences, Synthetic and Functional Biomolecules Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871 China
Institute of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, 200433 China;2. College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking-Tsinghua Center for Life Sciences, Synthetic and Functional Biomolecules Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871 China;3. Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS) and Institute of Computing Technology, CAS, Beijing, 100190 China;4. Institute of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, 200433 China Deseased |
| Abstract: | Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e., intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click-iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N-linked, mucin-type O-linked, and O-GlcNAcylated glycopeptides. We demonstrate the utility of Click-iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N-glycosites, 262 intact O-GalNAc glycosites, and 1947 O-GlcNAcylation sites were identified. Click-iG-enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways. |
| Keywords: | Glycoproteomics Glycosylation Intact Glycosites Metabolic Labeling Unnatural Glycans |