A Practical Experimental Protocol for Identification and Validation of UFMylation Substrate in Human Cells.

UFMylation is an evolutionarily conserved ubiquitin-like modification that covalently conjugates UFM1 to lysine residues of substrates via a sequential E1-E2-E3 enzymatic cascade. UFMylation plays a pivotal role in maintaining cellular homeostasis, and its dysregulation is closely linked to multiple major diseases, including malignant tumors, hematopoietic defects, neurodegenerative disorders, and congenital developmental defects, highlighting its important biological significance. However, few substrates of UFMylation have been reported to date, limiting our deep understanding of the mechanistic functions of this modification. This major bottleneck stems from two major technical limitations: the overwhelming abundance of ribosomal protein L26 (RPL26)-UFM1 conjugates masks signals from low-abundance substrates, and conventional methods rely on cumbersome cotransfection of multiple pathway components with poor efficiency and specificity in UFMylated peptides enrichment. To address these challenges, we have developed an effective and specific experimental protocol for UFMylation detection and large-scale substrate identification. This protocol employs CRISPR-Cas9-mediated gene editing to generate