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.

Rapid detection, discovery, and identification of post‐translationally myristoylated proteins during apoptosis using a bio‐orthogonal azidomyristate analog

“…This alternative method takes advantage of the Staudinger ligation's unique chemoselective reactivity that can covalently link alkyl azides, such as azidomyristate, to a tagged triarylphosphine via an amide bond, thereby allowing specific probing of azidomyristoylated proteins within the cell (15). Indeed, the fact that neither alkyl azides nor phosphines are found in the biological milieu and that they do not react with cellular nucleophiles at ambient temperatures makes them ideal for in vivo labeling and subsequent tagging of post-translationally modified proteins (15,16,27,28). Important to the biological study of myristoylation, 12-azidododecanoate is an alkyl azide analog of myristate that, like most alkyl azides, has been shown previously to be nontoxic to cells and animals (29).…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…This technique has been adapted for the detection and enrichment of O-GlcNAc-modified proteins and peptides. Sugars containing either ketones or azido groups are incorporated into O-GlcNAc-modified proteins using one of two techniques: (1) a mutant GalT1 (Y289L) with an enlarged active site is used to add UDP-GalNAz to terminal GlcNAc residues similar to the GalT1 labeling discussed above (Khidekel et al 2003); and (2) in cells by metabolic labeling with a peracetylated azido-GlcNAc substrate (Vocadlo et al 2003;Sprung et al 2005), as many of the enzymes in the HBP will tolerate unnatural sugars. However, the kinetics of O-GlcNAz removal from proteins by OGlcNAcase is significantly reduced.…”

Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry

“…One such example of this cross-disciplinary endeavor is the commandeering of cellular machinery to introduce synthetic tagged analogues of small molecule modifiers during co/post-translational modification. Notable examples of this have included glycosylation, 1-3 biotin ligation, 4-7 prenylation, [8][9][10] cholesterylation, 11 phosphorylation 12- 15 and lipidation.…”