An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• ADP-ribosylation is crucial for DNA repair, chromatin regulation, and RNA processing.
• Investigating ADP-ribosylation is challenging due to complex PAR chains, low modification abundance, and limited enrichment methods.

Purpose of the Study:

• To develop novel chemical probes for improved proteome-wide ADP-ribosylation analysis.
• To quantify cellular responses to PARP inhibitors using advanced mass spectrometry.

Main Methods:

• Utilized 2-alkyne adenosine (2YnAd), a clickable NAD+ precursor, for selective enrichment and detection of ADP-ribosylated proteins.
• Developed a dual metabolic labeling strategy combining 2YnAd and 6YnAd.
• Employed tandem mass tag (TMT) mass spectrometry and Bayesian analysis for quantitative proteomics.

Main Results:

• 2YnAd demonstrated superior protein coverage compared to 6YnAd in quantitative mass spectrometry.
• The dual labeling approach enabled global enrichment of ADP-ribosylated proteins.
• Successfully quantified thousands of protein responses to clinical PARP inhibitors Olaparib and Rucaparib.

Conclusions:

• 2YnAd is a valuable tool for sensitive and comprehensive ADP-ribosylation studies.
• The developed dual labeling and mass spectrometry strategy provides a powerful platform for drug response profiling.