Hundreds of nuclear, cytoplasmic, and mitochondrial proteins within multicellular eukaryotes have hydroxyl groups of specific serine and threonine residues modified by the monosaccharide Nacetylglucosamine (GlcNAc). This modification, known as O-GlcNAc, has emerged as a central regulator of both cell physiology and human health. A key emerging function of O-GlcNAc appears to be to regulate cellular protein homeostasis. We previously showed, using overexpressed model proteins, that O-GlcNAc

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... ication can occur co-translationally and that this process prevents premature degradation of such nascent polypeptide chains. Here we use tandem metabolic engineering strategies to label endogenously occurring nascent polypeptide chains within cells using O-propargyl-puromycin (OPP) and target the specific subset of nascent chains that are co-translationally glycosylated with O-GlcNAc by metabolic saccharide engineering using tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-D-galactopyranose (Ac4GalNAz). Using various combinations of sequential chemoselective ligation strategies we go on to tag these analytes with a series of labels, allowing us to define conditions that enable their robust labeling. Two-step enrichment of these glycosylated nascent chains, combined with shotgun proteomics, allows us to identify a set of endogenous co-translationally O-GlcNAc modified proteins. Using alternative targeted methods, we examine three of these identified proteins and further validate their co-translational O-GlcNAcylation. These findings detail strategies to enable isolation and identification of extremely low abundance endogenous analytes present within complex protein mixtures. Moreover, this work opens the way to studies directed at understanding the roles of O-GlcNAc and other co-translational protein modifications and should stimulate an improved understanding of the role of O-GlcNAc in cytoplasmic protein quality control and proteostasis.