Myristoylation profiling in human cells and zebrafish

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Protein lipidation, specifically myristoylation, is a crucial post-translational modification regulating protein function and localization.
• Understanding the dynamics of protein lipidation is essential for comprehending cellular processes and developmental biology.

Purpose of the Study:

• To identify and quantify co-translationally myristoylated proteins in human cells and zebrafish embryos.
• To develop and apply a novel chemical proteomics approach for studying protein lipidation.
• To investigate dynamic protein lipidation during vertebrate development.

Main Methods:

• Metabolic labeling of human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos with an alkynyl myristic acid probe.
• Cell lysis using SDS buffer followed by ligation of tagged proteomes to multifunctional capture reagents.
• Copper-catalyzed alkyne azide cycloaddition (CuAAC) for affinity enrichment.
• Mass spectrometry/mass spectrometry (MS/MS) for high-confidence identification of modification sites.

Main Results:

• Identification of 87 co-translationally myristoylated proteins in human cells.
• Identification of 61 co-translationally myristoylated proteins in zebrafish embryos.
• Direct MS/MS evidence confirmed modification sites for identified proteins.

Conclusions:

• The developed method enables quantitative, proteome-wide analysis of protein modifications.
• This study provides novel insights into the landscape of co-translational myristoylation in humans and vertebrates.
• The findings contribute to understanding dynamic protein lipidation in development.