LAUSR

Since the pioneering work by Reutter and co‐workers that demonstrated structural flexibility in the carbohydrate biosynthesis and glycosylation pathways, many different labs have used unnatural monosaccharide analogues to perform glycan engineering on the surface of living cells. A subset of these unnatural monosaccharides contain bioorthogonal groups that enable the selective installation of visualization or enrichment tags. These metabolic chemical reporters (MCRs) have proven to be powerful for the unbiased identification of glycoproteins; however, they do have certain limitations. For example, they are incorporated substoichiometrically into glycans, and most MCRs are not selective for one class (e.g., O‐GlcNAcylation) of glycoprotein. Here, we explore the relationship between the biosynthesis of MCR donor sugars in cells and the labeling levels of four different N‐acetylglucosamine‐ and N‐acetylgalactosamine‐based MCRs. We found that the buildup of the different donor sugars correlated well with the overall labeling levels but less so with intracellular labeling of proteins by O‐GlcNAcylation.