Photocrosslinking Sugars Capture Glycan‐Dependent Interactions

O‐linked N‐acetyl‐D‐glucosamine (O‐GlcNAc) is an abundant and dynamic single sugar post‐translational modification regulated by two enzymes, O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). Dysregulation of O‐GlcNAcylation is associated with human diseases, including cancer and diabetes. While hundreds of O‐GlcNAc‐modified proteins have been identified, less is known about how O‐GlcNAc‐ylation alters protein function. Identifying protein‐protein binding interactions that are promoted by O‐GlcNAc modification may provide insight into the functional consequences of O‐GlcNAc‐ylation. To achieve this, we prepare the “O‐GlcNDAz” modification, in which the diazirine photoactivable crosslinking group is appended onto O‐GlcNAc residues. Subsequent UV irradiation activates the diazirine, resulting in covalent crosslinks between O‐GlcNDAz‐modified proteins and neighboring molecules. The crosslinked complexes can be isolated and analyzed by mass spectrometry to identify binding partners. I will describe a chemoenzymatic method to prepare O‐GlcNDAz‐modified peptides in vitro, for subsequent crosslinking to proteins in lysates. In addition, I will describe improvements to a previously reported method to prepare O‐GlcNDAz in cells through metabolic engineering. Used together, the in vitro and cell‐based O‐GlcNDAz crosslinking methods provide insight into the functional roles of O‐GlcNAc modification. Support or Funding Information NIH (R01GM090271) and the Welch Foundation (I‐1686)Metabolism of photocrosslinking O‐GlcNAc (O‐GlcNDAz)