O-GlcNAc is a pleotropic, enigmatic post-translational modification (PTM). This PTM modifies thousands of proteins differentially across tissue types and regulates diverse cellular signaling processes. O-GlcNAc is implicated in numerous diseases, and the advent of O-GlcNAc perturbation as a novel class of therapeutic underscores the importance of identifying and quantifying the O-GlcNAc modified proteome. Here, we review recent advances in mass spectrometry-based proteomics that will be critical in elucidating the role of this unique glycosylation system in health and disease.

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Activation of ERK (Extra‐cellular Regulated Kinase) signal plays a major role in metabolic diseases such as cancer and Alzheimer's. Ingenuity Pathway Analysis (IPA) of RNA‐Seq data from long‐term O‐GlcNAcase (OGA) enzyme inhibitor‐ Thiamet‐G (TMG) treated SH‐SY5Y (neuroblastoma) cells, which elevates O‐GlcNAc levels, indicated ERK Signaling as a top upregulated pathway. O‐GlcNAcylation consists of the addition of a single N‐acetyl‐glucosamine residue (GlcNAc) to specific serine/threonine residues of proteins by the enzyme OGT (O‐GlcNAc Transferase) and the removal of O‐GlcNAc is catalyzed by the enzyme OGA. O‐GlcNAc is highly abundant and is known to modulate kinase activity. Thus, to further investigate the mechanism by which O‐GlcNAcylation activates ERK, we did a serum reactivation time‐course and found that there is an amplification in ERK phosphorylation after long‐term TMG treatment in HeLa and SH‐SY5Y cells. Next, TGF‐β stimulation that specifically activates ERK signaling also indicated an increase in ERK phosphorylation with long‐term TMG treatment, confirming a robust ERK activation with TMG treatment. ERK phosphorylation oscillates with time after mitogen activation; therefore, we wanted to see if TMG treatment influenced ERK oscillation for a period of 24 hours. Serum reactivation revealed an oscillation in phosphorylated‐ERK expression within a 24‐hour period, where it reaches a maximum at 4 hours and slowly decreases at 8,12 and 24 hours upon long‐term TMG treatment. Next, we wanted to further evaluate the mechanism by which TMG treatment increases ERK phosphorylation. Of note, we did not measure any O‐GlcNAcylation on ERK; hence, we probed the expression of activated mitogen activated protein kinase‐kinase (phosphorylated MEK) which phosphorylates ERK and Dual specificity phosphatases (DUSPs), which dephosphorylate ERK. Interestingly, serum reactivation time course showed an increase in both phosphorylated‐MEK and total DUSP4 with TMG treatment. In addition to the use of pharmacological OGA inhibitor, we measured the effect of OGT knock‐down (KD) and OGA KD on ERK signaling in SH‐SY5Y and HeLa cells. There was amplification in ERK phosphorylation even after OGT knock‐down (OGT KD) which lowers the level of O‐GlcNAcylation, in contrast to long‐term TMG treatment which increases O‐GlcNAcylation. These data suggest that the cycling of the modification on and off substrates is key to ERK pathway regulation. Recent research also shows APOE4 stimulates the transcription of amyloid precursor protein (APP) via a non‐canonical ERK signaling pathway, leading to increased amyloid‐beta secretion in Alzheimer's disease pathogenesis. Therefore, we wanted to see if there is an increase in the expression of APP in the long‐term TMG treated SH‐SY5Y cells after serum reactivation, and we saw an increase in APP expression correlating with amplification of ERK signaling. Further evaluation of these molecular mechanisms to elucidate how O‐GlcNAcylation amplifies ERK signaling and understand if O‐GlcNAcylation and ERK a...