TY  - JOUR
TI  - A Practical Experimental Protocol for Identification and Validation of UFMylation Substrate in Human Cells
AU  - Liang, Qian 
AU  - Fang, Yaoyao 
AU  - Dong, Juexi 
AU  - Yi, Xingling 
AU  - Cong, Yu-Sheng 
VL  - 16
IS  - 14
PY  - 2026
DA  - 2026/07/20
SP  - e5758
C1  - Bio-protocol 2026;16:e5758
DO  - 10.21769/BioProtoc.5758
UR  - https://doi.org/10.21769/BioProtoc.5758
AB  - 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 UFSP1/UFSP2 double-knockout (UFSP1KO/UFSP2KO, DKO) HEK293T cells, which completely abrogate de-UFMylation and thus significantly elevate global protein UFMylation levels upon exogenous introduction of mature UFM1-ΔC2. In addition, exogenous co-expression of the E3 ligase core components UFL1 and DDRGK1 can further improve the sensitivity of substrate detection. This protocol enables large-scale identification of UFMylation substrates with modification sites via high-efficiency enrichment with the K-ε-VG antibody and LC-MS/MS analysis.
KW  - UFMylation
KW  - Substrate
KW  - UFSP1/2
KW  - UFL1
KW  - DDRGK1
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False