An Optimized Isotopic Photocleavable Tagging Strategy
for Site-Specific and Quantitative Profiling of Protein O‑GlcNAcylation
in Colorectal Cancer Metastasis
posted on 2022-03-07, 15:05authored byJialin Liu, Yi Hao, Chunting Wang, Yangya’nan Jin, Yong Yang, Jin Gu, Xing Chen
O-linked-β-N-acetylglucosamine (O-GlcNAc)
glycosylation is a ubiquitous protein post-translational modification
of the emerging importance in metazoans. Of the thousands of O-GlcNAcylated
proteins identified, many carry multiple modification sites with varied
stoichiometry. To better match the scale of O-GlcNAc sites and their
dynamic nature, we herein report an optimized strategy, termed isotopic
photocleavable tagging for O-GlcNAc profiling (isoPTOP), which enables
quantitative and site-specific profiling of O-GlcNAcylation with excellent
specificity and sensitivity. In HeLa cells, ∼1500 O-GlcNAcylation
sites were identified with the optimized procedures, which led to
quantification of ∼1000 O-GlcNAcylation sites with isoPTOP.
Furthermore, we apply isoPTOP to probe the O-GlcNAcylation dynamics
in a pair of colorectal cancer (CRC) cell lines, SW480 and SW620 cells,
which represent primary carcinoma and metastatic cells, representatively.
The stoichiometric differences of 625 O-GlcNAcylation sites are quantified.
Of these quantified sites, many occur on important regulators involved
in tumor progression and metastasis. Our results provide a valuable
database for understanding the functional role of O-GlcNAc in CRC.
IsoPTOP should be applicable for investigating O-GlcNAcylation dynamics
in various pathophysiological processes.