Application of a Novel Myristoylproteomics Approach Identifies GLIPR2 as a Key Pro-Ferroptotic Substrate in Non-Small Cell Lung Cancer

Protein myristoylation, catalyzed by N-myristoyltransferases (NMT1 and NMT2), is a key co- and post-translational modification involved in cellular signaling, yet its role in ferroptosis remains poorly defined. Here, we developed a novel myristoylproteomics workflow leveraging click chemistry to comprehensively profile N-myristoylated proteins within NSCLC cells. We found that NMT1 and NMT2 expression positively correlates with ferroptosis sensitivity. Genetic or pharmacological inhibition of NMT attenuated ferroptosis, whereas their overexpression enhanced it. Using our optimized quantitative myristoylproteomics platform, we identified GLIPR2 as a novel myristoylated protein with elevated modification levels in ferroptosis-sensitive cells. Functional studies confirmed that GLIPR2 promotes ferroptosis, and this pro-ferroptotic activity of GLIPR2 requires its N-myristoylation, as a myristoylation-deficient mutant (G2A) failed to restore ferroptosis sensitivity. Our work establishes a robust proteomic methodology for mapping protein myristoylation and uncovers the NMT1/NMT2–GLIPR2 axis as a pivotal regulator of ferroptosis. These findings propose new therapeutic strategies for NSCLC. • Developed and optimized a novel click chemistry-based myristoylproteomics approach for systematic identification of NMT substrates. • Discovered that NMT1/NMT2 expression positively correlates with ferroptosis sensitivity in NSCLC cells. • Screened out GLIPR2 as a new myristoylated protein (higher modification in ferroptosis-sensitive cells) via this technique. • Functional validation showed GLIPR2 exerts pro-ferroptotic effect depending on its myristoylation modification.