Mass Spectrometry of Glycoproteins

Volume 951 of the series Methods in Molecular Biology pp 57-67


Incorporation of Unnatural Sugars for the Identification of Glycoproteins

  • Balyn W. ZaroAffiliated withDepartment of Chemistry, University of Southern California
  • , Howard C. HangAffiliated withThe Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University
  • , Matthew R. PrattAffiliated withDepartment of Chemistry, University of Southern CaliforniaDepartment of Molecular and Computational Biology, University of Southern California Email author 

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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 words

Proteomics Glycosylation Bioorthogonal chemical reporter Click chemistry Azide Mass spectrometry O-GlcNAc