A hydroxylamine probe for profiling S-acylated fatty acids on proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Reversible S-palmitoylation regulates protein function and localization.
• Protein S-acylation extends beyond palmitate to include various fatty acids.
• The full diversity of protein acylation remains underexplored.

Purpose of the Study:

• To develop a chemical probe for detecting and quantifying diverse protein-linked fatty acids.
• To profile the S-acylome using this novel chemical probe.
• To investigate the acylation heterogeneity of the oncogene N-Ras.

Main Methods:

• Development of a chemical probe for fatty acid detection.
• Mass spectrometry (MS)-based analysis for protein acylation profiling.
• Utilizing semisynthetic proteins to study membrane subdomain distribution.

Main Results:

• A chemical probe enabling rapid detection and quantification of protein-linked fatty acids was developed.
• The S-acylome was profiled, revealing heterogeneous acylation of N-Ras.
• N-Ras was shown to be acylated with both palmitate and palmitoleate.
• Unsaturated N-Ras exhibited increased membrane subdomain clustering and faster insertion kinetics.

Conclusions:

• The developed chemical probe facilitates comprehensive S-acylome analysis.
• N-Ras displays heterogeneous acylation with distinct fatty acids, influencing its membrane dynamics.
• Understanding diverse protein acylation is crucial for protein function and localization studies.