title: Tagging-via-substrate strategy for probing O-GlcNAc modified proteins.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Sprung
authors.forename: Animesh
authors.initials: A
authors.lastname: Nandi
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
authors.forename: Sung Chan
authors.initials: SC
authors.lastname: Kim
authors.forename: Deb
authors.initials: D
authors.lastname: Barma
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: Yingming
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000117:Acetylglucosamine; D000818:Animals; D001386:Azides; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D015336:Molecular Probe Techniques; D010766:Phosphorylation; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*
chemical_list: D001386:Azides; D006023:Glycoproteins; D008024:Ligands; D000117:Acetylglucosamine