Tagging-via-Substrate Strategy for Probing O-GlcNAc Modified Proteins Robert Sprung, † Animesh Nandi, † Yue Chen, † Sung Chan Kim, † Deb Barma, † John R. Falck, †,‡ and Yingming Zhao* ,†,‡, Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038 Received February 14, 2005 Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic analysis of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, we report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves three steps: metabolic labeling of cells with an unnatural GlcNAc analogue, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purification of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymatic systems among these cells from diverse biological species. We isolated azido-GlcNAc modified proteins from the cytosolic extract of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic analysis of O-GlcNAc modified proteins. Keywords: glycosylation • O-GlcNAc • proteomics • tagging-via-substrate • Staudinger ligation • post-translational modifications Introduction A major goal of proteomics studies is the global analysis of the interactions, expression, and modifications of the diverse and dynamic cellular proteins and to correlate this information with protein function and cellular regulation. Unfortunately, due to the wide dynamic range and high complexity of a cell’s proteome, the global profiling of low-to-medium abundance proteins and post-translational modifications is impractical. More powerful separation and mass spectrometry tools are necessary to deal with this daunting challenge. One strategy to address the problems is to develop robust technologies for the selective detection and isolation of proteins bearing specific modifications. The O-GlcNAc modification, an abundant post-translational modification present at serine and threonine residues of nuclear and cytosolic proteins, was discovered by Hart and his colleagues about two decades ago. 1 The modification is found in all higher eukaryotes from C. elegans to mammals and is dynamic, possessing the ability to respond to external stimuli. 2-6 The modification has been shown to be involved in the modulation of protein-protein interactions, protein-DNA bind- ing, protein localizations, and protein stability. 3 The fast turnover of O-GlcNAc is the result of the interplay between O-GlcNAc transferase (OGT), 7 which adds GlcNAc to proteins, and the O-GlcNAc-specific -N-acetylglucosaminidase (O- GlcNAcase), 8 which removes it. Both enzymes are ubiquitously expressed in mammalian cells and OGT has been shown to be essential for cell viability. 9 An important consideration with regard to the O-GlcNAc modification is that the intracellular level of UDP-GlcNAc, the substrate for GlcNAc transfer reaction, is regulated by glucose. It is estimated that between 2 and 5% of the glucose taken up by a cell is diverted through the hexosamine biosynthetic pathway leading to formation of UDP-GlcNAc. 10 Thus, the position of UDP-GlcNAc in the hexosamine biosynthetic pathway, coupled with the rapid turnover of O-GlcNAc, im- plicates the O-GlcNAc modification as an energy sensor and regulatory modification. 3 Indeed, dysregulation of the O- GlcNAc modification has been shown to be involved in the development of insulin resistance and diabetes. 11-13 * To whom correspondence should be addressed. Tel: (214) 648-7947. Fax: (214) 648-2797. E-mail: yzhao@biochem.swmed.edu. † Departments of Biochemistry. ‡ Pharmacology. 950 Journal of Proteome Research 2005, 4, 950-957 10.1021/pr050033j CCC: $30.25  2005 American Chemical Society Published on Web 05/20/2005