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  1. Home
  3. Activity-based Protein Profiling For Functional Cysteines And Protein Target Identification.
  1. Home
  3. Activity-based Protein Profiling For Functional Cysteines And Protein Target Identification.

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Activity-Based Protein Profiling for Functional Cysteines and Protein Target Identification.

Tin-Yan Koo1, Clive Yik-Sham Chung2,3

  • 1School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, People's Republic of China.

Methods in Molecular Biology (Clifton, N.J.)
|June 14, 2025

View abstract on PubMed

Summary
This summary is machine-generated.

This study introduces NAIA, a novel acrylamide probe for activity-based protein profiling (ABPP). NAIA enhances cysteine reactivity for improved functional cysteine identification and covalent ligand target discovery in cancer research.

Keywords:
AcrylamideActivity-based probesActivity-based protein profilingChemoproteomicsCysteine profilingCysteinomeMass spectrometryTarget identification

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Proteomics

Background:

  • Activity-based protein profiling (ABPP) is crucial for studying functional cysteines.
  • Competitive ABPP aids in discovering lead compounds and identifying their protein targets.
  • Cysteine residues play vital roles in cellular functions and disease.

Purpose of the Study:

  • To present a detailed protocol for using the novel NAIA probe in mass spectrometry-based ABPP.
  • To profile functional cysteines within cancer cells and cell lysates.
  • To identify protein targets of covalent ligands using the NAIA workflow.

Main Methods:

  • Utilized a new class of acrylamide-based cysteine probe, NAIA, with enhanced reaction kinetics.
  • Employed mass spectrometry-based activity-based protein profiling (MS-ABPP).
  • Developed a step-by-step protocol for applying NAIA in complex biological samples.

    • Main Results:

    • NAIA demonstrated superior cysteine reaction kinetics compared to existing probes.
    • The protocol effectively enabled proteome-wide cysteine profiling in cancer cell contexts.
    • Successful application of NAIA for identifying protein targets of covalent ligands was achieved.

    Conclusions:

    • NAIA represents an advanced tool for comprehensive functional cysteine analysis.
    • The described MS-ABPP protocol facilitates robust target identification for covalent inhibitors.
    • This methodology advances the study of cysteine-mediated biological processes and drug discovery.