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

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.