Tang_2026_Crit.Rev.Anal.Chem__1

Protein S-palmitoylation is a reversible protein post-translational lipid modification that dynamically regulates protein-membrane affinity and subcellular localization, playing crucial roles in cellular signaling, metabolic regulation, immune responses, and other physiological processes. Therefore, accurate detection and quantification of these modified proteins are essential for elucidating their biological functions and involvements in disease mechanisms. Conventional techniques such as acyl-biotin exchange (ABE) and metabolic labeling have enabled the discovery and study of S-palmitoylation in biological samples. However, these methods still face challenges including low sensitivity, limited throughput, and difficulties in multiplexed detection. This review systematically outlines the biochemical mechanisms of S-palmitoylation and depalmitoylation, summarizes established methods for substrate modification detection and catalytic enzyme activity assays, and analyzes challenges in the field, including enzyme isoform specificity, tool development, and application in complex biological systems. Finally, we highlight emerging strategies aimed at achieving real-time, spatially resolved, and clinically translatable detection. Continued innovation in this field is poised to deepen our understanding of S-palmitoylation dynamics and accelerate the development of targeted therapies for related diseases.