Deciphering the Functional O‐GlcNAc Sub‐Proteome

O‐linked N‐acetyl‐β‐D‐glucosamine (O‐GlcNAc) is a post‐translational modification of serine and threonine residues that regulates protein function and signaling. O‐GlcNAc has emerged as a novel regulator of the cellular response to stress, including oxidative stress and ischemia reperfusion (IR) injury of the heart. A recent study has identified over 200 proteins whose state of O‐GlcNAcylation changes in response to oxidative stress. However, the specific pathways that regulate this response remain ill‐defined. This is in‐part due to the promiscuous nature of proteins to associate in complexes in order to mediate cellular pathways. Thus, the goal of this study is to characterize the O‐GlcNAc modified protein complexes that change in response to oxidative stress. Proteins were isolated from mouse embryonic fibroblasts, followed by affinity enrichment for O‐GlcNAcylated protein complexes. Complexes were then separated by size exclusion chromatography (SEC). Preliminary data demonstrates differential elution of O‐GlcNAc modified proteins in distinct SEC fractions. In addition, known O‐GlcNAc modified proteins such as CARM1 elute in the same SEC fractions as known binding proteins, FUS and HNRNPU. Together these data support the use of affinity enrichment coupled to SEC for the characterization of O‐GlcNAc modified protein complexes. Current studies are now focused on the identification of proteins within complexes by mass spectrometry with the ultimate goal of quantification by stable isotopic labeling of amino acids in cell culture. Together, these data will provide a more detailed understanding of the role of O‐GlcNAc in response to oxidative stress and IR injury.