Protein fatty-acylation in eukaryotes has been associated with many fundamental biological processes. However, the diversity, abundance and regulatory mechanisms of protein fatty-acylation in vivo remain to be explored. Herein, we review the proteomic analysis of fatty-acylated proteins, with a focus on N-myristoylation and S-palmitoylation. We then highlight major challenges and emerging methods for direct site identification, quantitation, and lipid structure characterization to understand the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.
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