An Isotope-Coded Photocleavable Probe for Quantitative Profiling of Protein O-GlcNAcylation

Area of Science:

• Biochemistry
• Proteomics
• Cell Biology

Background:

• O-linked N-acetylglucosamine (O-GlcNAc) is a crucial post-translational modification impacting cell function.
• Understanding O-GlcNAcylation dynamics at the proteome-wide level is vital for elucidating cellular mechanisms and roles.

Purpose of the Study:

• To develop and validate an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics.
• To enable quantitative mass spectrometry-based proteomics for O-GlcNAcylation analysis.

Main Methods:

• Development of a novel isotope-coded photocleavable probe.
• One-step selective tagging and isotopic labeling of O-GlcNAcylated proteins from complex mixtures.
• Quantitative mass spectrometry-based proteomics.

Main Results:

• The probe successfully enabled quantitative profiling of O-GlcNAcylation sites in 293T cells.
• The probe was applied to analyze O-GlcNAcylation stoichiometry in liver cancer cells with varying sorafenib sensitivity.
• Demonstrated the probe's utility in investigating O-GlcNAcylation's role in cancer chemoresistance.

Conclusions:

• The developed probe is a powerful tool for profiling protein O-GlcNAcylation dynamics.
• This method provides a foundation for mechanistic investigations into O-GlcNAcylation in cellular processes, including cancer chemoresistance.