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A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
Published on: March 13, 2014
Chemical proteomic approaches to investigate N-myristoylation
James Zhang1, Wouter W Kallemeijn2, Sarah Hassan3
1Department of Chemistry, Imperial College London, London, United Kingdom; The Francis Crick Institute, London, United Kingdom; Division of Clinical Studies, The Institute of Cancer Research (ICR) & Royal Marsden NHS Trust, London, United Kingdom.
This study details two chemical proteomic methods to analyze N-myristoylation, a crucial protein lipidation process. These techniques enable researchers to study N-myristoylation in various biological contexts and in response to drug treatments.
Area of Science:
- Biochemistry
- Proteomics
- Chemical Biology
Background:
- N-myristoylation is a key protein lipidation catalyzed by N-myristoyltransferase (NMT) enzymes, affecting over 200 proteins with N-terminal glycines.
- This modification regulates protein localization, stability, function, and interactions, playing a role in biological processes and diseases.
- Understanding N-myristoylation changes in disease and during NMT inhibition is vital for basic biology and clinical applications.
Purpose of the Study:
- To describe two complementary chemical proteomic methods for assessing N-myristoylation.
- To provide detailed protocols for these methods, aiding research into N-myristoylation's role in biology and disease.
Main Methods:
- Metabolic labeling: Incorporating bio-orthogonal myristic acid analogs into NMT substrates for selective labeling, enrichment, and detection.
- Sortase A-mediated labeling: Using Sortase A to attach an enrichment handle to NMT substrates, particularly useful when NMT activity is inhibited.
Main Results:
- The chapter presents detailed protocols for both metabolic labeling and Sortase A-based chemical proteomic approaches.
- These methods allow for the selective labeling, enrichment, and detection of N-myristoylated proteins.
- The protocols cover cell treatment, sample preparation, and data analysis for comprehensive N-myristoylation profiling.
Conclusions:
- Two robust chemical proteomic methods are presented for profiling the N-myristoylated proteome.
- These techniques are valuable for investigating N-myristoylation in health, disease, and response to NMT inhibitors.
- The provided protocols facilitate advancements in understanding N-myristoylation's biological roles and therapeutic potential.
