Ac₃4deoGlcNAz: A Selective Probe for Identifying O-GlcNAc-Modified Proteins in Prostate Cancer

GlcNAcylation is a prevalent protein modification in eukaryotic cells and increasing evidences indicated that over-expressed O-GlcNAcylation are intimately linked to the development and prognosis of prostate cancer. Thus, exploring this modification in the context of prostate cancer is vital for understanding the underlying mechanisms and hopefully used for future targeted therapies. In this paper, we use our previously established metabolic probes to comprehensively compare the labeling efficiency of O-GlcNAc modified proteins in PC3 cells. Our results demonstrated that all the tested probes were non-toxic to PC3 cells and only Ac₄GlcNAz, Ac₄GalNAz and Ac₃4deoGlcNAz exhibited robust labeling signals amongst the probes. Further investigations by western blot and flow cytometry analysis revealed that Ac₃4deoGlcNAz was a specific and efficient probe for intracelluar protein labeling with negligible S-glyco-modification signal. In addition, proteomic analysis further confirmed that 94 % of the proteins identified by Ac₃4deoGlcNAz were in the form of O-linked GlcNAc rather than S-glyco-modification, these enriched O-GlcNAcylated proteins were mainly involved in the regulated processes of prostate cancer. Our results here together prove Ac₃4deoGlcNAz is a safe and reliable probe for metabolic labeling O-GlcNAc modified proteins in prostate cancer, providing a mean to fully exploit the regulatory mechanism of O-GlcNAcylation in the process of prostate cancer.