Citation: Pengfei Wu, Chang Sun, Xiao Huang, Wenjing Nie, Qiongqiong Wan, Qingtao Meng, Suming Chen. Dual-identification strategy for global profiling of protein fatty acylation sites[J]. Chinese Chemical Letters, ;2026, 37(10): 112530. doi: 10.1016/j.cclet.2026.112530 shu

Dual-identification strategy for global profiling of protein fatty acylation sites

The Institute for Advanced Studies, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430072, China

qtmeng@whu.edu.cn

sm.chen@whu.edu.cn

Received Date: 28 October 2025
Revised Date: 23 January 2026
Accepted Date: 13 February 2026
Available Online: 14 February 2026

Figures(5)

Protein fatty acylation encompasses S/N/O-acylation modifications that regulate diverse cellular functions, yet current methods cannot simultaneously capture all three types. Here, we present a dual-identification strategy that integrates metabolic labeling with click chemistry, hydroxylamine mediated selective hydrolysis, and cleavable bioorthogonal probes for comprehensive fatty acylation site detection. Using Alk-C16:0 and Alk-C18:0 probes in HepG2 cells, we identified 1310 and 1131 total fatty acylation sites respectively, including 588 and 674 S-acylation sites substantially exceeding single method coverage. Integration of open and closed searches revealed probe intracellular conversion induced acyl chain heterogeneity, improving identification accuracy. We discovered diagnostic ions revealing acyl chain structure and N-acylation specific cyclic immonium ions that discriminate between modification subtypes. This strategy enables unprecedented comprehensive protein fatty acylation characterization, providing a robust platform for investigating molecular mechanisms of these essential modifications.
- Fatty acylation,
- Mass spectrometry,
- Metabolic labeling,
- Click chemistry,
- Palmitoylation
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