"Two Birds One Stone" Strategy for the Site-Specific Analysis of Core Fucosylation and O-GlcNAcylation

  • J Am Chem Soc. 2023 Jul 26;145(29):15879-15887. doi: 10.1021/jacs.3c02976.
Yawen Luo  1  2 ,  Yuqiu Wang  3  4 ,  Yinping Tian  1 ,  Hu Zhou  3  2 ,  Liuqing Wen  1  5  2
Affiliations
  • 1. Carbohydrate-Based Drug Research Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
  • 2. University of Chinese Academy of Sciences, Beijing 100049, China.
  • 3. CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
  • 4. School of Bioengineering, East China University of Science and Technology, Shanghai 200237, China.
  • 5. State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
PMID: 37340703 DOI: 10.1021/jacs.3c02976
Abstract

Core fucosylation and O-GlcNAcylation are the two most famous protein glycosylation modifications that regulate diverse physiological and pathological processes in living organisms. Here, a "two birds one stone" strategy has been described for the site-specific analysis of core fucosylation and O-GlcNAcylation. Taking advantage of two mutant endoglycosidases (EndoF3-D165A and EndoCC-N180H), which efficiently and specifically recognize core fucose and O-GlcNAc, glycopeptides can be labeled using a biantennary N-glycan probe bearing azido and oxazoline groups. Then, a temperature-sensitive poly(N-isopropylacrylamide) polymer functionalized with dibenzocyclooctyne was introduced to facilitate the enrichment of the labeled glycopeptides from the complex mixture. The captured glycopeptides can be further released enzymatically by wild-type endoglycosidases (EndoF3 and EndoCC) in a traceless manner for mass spectrometry (MS) analysis. The described strategy allows simultaneous profiling of core-fucosylated glycoproteome and O-GlcNAcylated glycoproteome from one complex sample by MS technology and searching the database using different variable modifications.

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