Quantitative time-resolved chemoproteomics reveals that stable -GlcNAc regulates box C/D snoRNP biogenesis.

-linked GlcNAcylation (-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of -GlcNAcylated proteins, we developed a quantitative time-resolved -linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an -GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 -GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of -GlcNAc or degradation of protein backbones. The stability of those hyperstable -GlcNAcylated proteins was more sensitive to -GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that -GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking -GlcNAcylation on FBL altered the 2'--methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.