Incorporation of Unnatural Sugars for the Identification of Glycoproteins
Glycosylation is an abundant post-translational modification that alters the fate and function of its substrate proteins. To aid in understanding the significance of protein glycosylation, identification of target proteins is key. As with all proteomics experiments, mass spectrometry has been established as the desired method for substrate identification. However, these approaches require selective enrichment and purification of modified proteins. Chemical reporters in combination with bioorthogonal reactions have emerged as robust tools for identifying post-translational modifications including glycosylation. We provide here a method for the use of bioorthogonal chemical reporters for isolation and identification of glycosylated proteins. More specifically, this protocol is a representative procedure from our own work using an alkyne-bearing O-GlcNAc chemical reporter (GlcNAlk) and a chemically cleavable azido-azo-biotin probe for the identification of O-GlcNAc-modified proteins.
Key wordsProteomics Glycosylation Bioorthogonal chemical reporter Click chemistry Azide Mass spectrometry O-GlcNAc
We thank Leslie Bateman for careful reading of the manuscript. This work was supported by the University of Southern California and the American Cancer Society Grant IRG-58-007-51 (to M.R.P.) and the Ellison Medical Foundation and the National Institutes of Health and General Medical Sciences Grant 1RO1GM087544 O1A2 (to H.C.H.).
- 7.Zaro BW, Bateman LA, Pratt MR (2011) Robust in-gel fluorescence detection of mucin-type O-linked glycosylation. Bioorg Med Chem Lett 21(17):5062–5066Google Scholar
- 10.Zaro BW, Yang Y-Y, Hang HC, Pratt MR (2011) Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108(20):8146–8151Google Scholar
- 15.Wang Z et al (2010) Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Science STKE 3(104):ra2Google Scholar