A Practical Experimental Protocol for Identification and Validation of UFMylation Substrate in Human Cells
人细胞中UFM1修饰底物的鉴定与验证实验方案
(§Technical contact: lq@hznu.edu.cn) 发布: 2026年07月20日第16卷第14期 DOI: 10.21769/BioProtoc.5758 浏览次数: 1125
评审: Catherine HurdAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2026
Abstract
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.
Key features
• Employs UFSP1/UFSP2 DKO HEK293T cells with exogenous mature UFM1-ΔC2 to enhance UFMylation.
• Simplified transfection via single-factor assays using UFM1-ΔC2, UFL1, and DDRGK1.
• Combines K-ε-VG antibody enrichment with LC-MS/MS for large-scale substrate identification.
• Verifies UFMylation sites via site-directed mutagenesis and UFSP2-mediated de-UFMylation.
Keywords: UFMylation (UFM1修饰)
Substrate (底物)
UFSP1/2 (UFSP1/2)
UFL1 (UFL1)
DDRGK1 (DDRGK1)
Graphical overview
Graphical overview of the workflow for identifying and validating UFMylation substrates in human cells. This graphical overview summarizes the key steps of the optimized protocol. (A) Large-scale substrate identification by LC-MS/MS: Performed in UFSP1/UFSP2 double-knockout (DKO) HEK293T cells, followed by proteomic identification of UFMylated substrates via LC-MS/MS. (B) Validation of exogenously expressed substrates: Performed in DKO HEK293T cells, using immunoprecipitation (IP) and western blotting to confirm substrate UFMylation. (C) Endogenous substrate UFMylation assay: Conducted in wild-type (WT) HEK293T cells, to detect endogenous UFMylation via immunoprecipitation (IP) and western blotting.
Background
UFMylation is an evolutionarily conserved ubiquitin-like post-translational modification, in which ubiquitin-fold modifier 1 (UFM1) is covalently attached to lysine residues of target substrates through the sequential catalytic cascade involving three core components: the E1 activating enzyme UBA5, the E2 conjugating enzyme UFC1, and the multi-subunit E3 ligase complex comprising UFL1, UFBP1, and CDK5RAP3 [1–3]. Aberrant UFMylation is closely associated with cancers, neurodevelopmental disorders, and skeletal dysplasia [1,4,5].
In human cells, UFM1 is initially synthesized as an 85-amino-acid precursor (pro-UFM1). Its C-terminal Ser-Cys dipeptide is proteolytically trimmed by UFM1-specific proteases (UFSPs) to generate the mature 83-residue UFM1 variant termed UFM1-ΔC2, a truncated form competent for subsequent substrate conjugation [6]. Reversible UFMylation homeostasis is tightly regulated by two UFSP isoforms: UFSP1 mainly mediates pro-UFM1 maturation, whereas endoplasmic reticulum (ER)-resident UFSP2 predominantly catalyzes de-UFMylation to cleave conjugated UFM1 from modified substrates [7,8]. Conventional UFMylation detection methods require cumbersome cotransfection of multiple core components and suffer from low efficiency; meanwhile, the dominant high-abundance RPL26-UFM1 conjugates mask signals of low-abundance substrates, resulting in fewer than 30 verified UFMylation substrates to date, greatly hindering mechanistic studies [9,10]. Additionally, the shortage of modification-specific antibodies capable of capturing UFMylated peptides has long impeded high-throughput proteome-wide identification of UFMylation sites. The K-ε-VG antibody is a modification-specific affinity antibody raised against the remnant K-ε-VG epitope retained on substrates after proteolytic digestion; this reagent enables specific enrichment of UFM1-modified peptides and serves as the core tool for large-scale UFMylation site mapping via liquid chromatography–tandem mass spectrometry (LC-MS/MS) [11].
This optimized protocol employs UFSP1/UFSP2 double-knockout HEK293T cells combined with exogenous mature UFM1-ΔC2 to boost UFMylation and simplifies transfection by only using UFM1-ΔC2, UFL1, and DDRGK1 through single-factor optimization. It integrates K-ε-VG antibody enrichment and LC-MS/MS for high-throughput substrate identification, and validates modification sites via site-directed mutagenesis and UFSP2-mediated de-UFMylation assays, enabling the identification of over 600 UFMylation substrates with high specificity and efficiency [11].
This protocol has three minor limitations relevant to experimental execution: (1) It is primarily validated in HEK293T cells, (2) it has limited sensitivity for extremely low-abundance substrates, and (3) overexpression of modification components may introduce minor stoichiometric artifacts. A detailed mechanistic discussion is available in the Limitations of the study section of our accompanying JBC article [11]. Beyond substrate identification, this protocol can be widely applied to verify UFMylation sites, explore regulatory mechanisms of the UFMylation system, and screen disease-related UFMylation substrates in diverse mammalian cell models.
Materials and reagents
Biological materials
1. HEK293T cell line (ATCC, catalog number: CRL-3216)
2. UFSP1KO/UFSP2KO-HEK293T cell line was generated as a single-cell clone from parental HEK293T cells (CRL-3216) via CRISPR-Cas9-mediated genome editing and validated by Sanger sequencing. This cell line is available upon reasonable request from the corresponding author. Alternatively, users may generate this cell line independently following the detailed CRISPR-Cas9 editing protocol described in our previous publication [7].
Reagents
1. Polyethylenimine (PEI) (Sigma-Aldrich, catalog number: 765090-1G)
2. Na2HPO4·12H2O (Sigma-Aldrich, CAS number: 10039-32-4)
3. KH2PO4 (Sigma-Aldrich, CAS number: 7778-77-0)
4. Tris-HCl (1 M, pH 6.8) (Beyotime Biotechnology, catalog number: ST768)
5. Tris-HCl (1 M, pH 7.4) (Beyotime Biotechnology, catalog number: ST774)
6. Tris-HCl (1 M, pH 8.0) (Beyotime Biotechnology, catalog number: ST780)
7. Sodium dodecyl sulfate (SDS) (BioFroxx, catalog number: 3250GR500)
8. Glycerol (Sangon Biotech, CAS number: 56-81-5)
9. Dithiothreitol (DTT) (Macklin, catalog number: D806827)
10. Bromophenol blue (Diamond, CAS number: 115-39-9)
11. NaCl (Sinopharm, CAS number: 7647-14-5)
12. Nonidet P-40 (NP-40) (Biosharp, catalog number: BS205)
13. EDTA (0.5 M, pH 8.0) (Beyotime Biotechnology, catalog number: C0196)
14. Tris base (BioFroxx, catalog number: 1115KG001)
15. Glycine (Sinopharm, CAS number: 56-40-6)
16. Methanol (Sinopharm, CAS number: 67-56-1)
17. KCl (Sinopharm, CAS number: 7447-40-7)
18. Iodoacetamide (BBI Life Sciences, CAS number: 144-48-9)
19. Acetone (Sinopharm, CAS number: 67-64-1)
20. Triethylammonium bicarbonate (Sigma-Aldrich, catalog number: T7408)
21. CaCl2·2H2O (Sigma-Aldrich, CAS number: 10035-04-8)
22. Trypsin Gold, mass spectrometry grade (Promega, catalog number: V5280)
23. Trifluoroacetic acid (TFA) (Sigma-Aldrich, CAS number: 76-05-1)
24. Tween-20 (BioFroxx, catalog number: 1247LT001)
25. Nonfat powdered milk (EpiZyme Biotechnology, catalog number: PS112L)
26. ProClean 950 (Beyotime Biotechnology, catalog number: ST855)
27. Bovine serum albumin (BSA) (BioFroxx, catalog number: 4240GR500)
28. DMEM with high glucose (Viva Cell, catalog number: C3113-0500)
29. Fetal bovine serum (FBS) (ExCell, catalog number: FSP500)
30. Penicillin-streptomycin solution (100×) (Biological Industries, catalog number: 03-031-1B)
31. Trypsin-EDTA solution (Biological Industries, catalog number: 03-050-1BCS)
32. Opti-MEM (Gibco, catalog number: 31985-070)
33. MG132 (Selleck Chemicals, catalog number: S2619)
34. Anti-FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220)
Note: Storage: -20 °C. Shelf life: ≥6 months unopened. Handling: Centrifuge briefly at 500× g for 30 s to collect beads before use; do not freeze-thaw the resin repeatedly.
35. Protein A/G agarose (Proteintech, catalog number: PR40025)
Note: Storage: 4 °C. Shelf life: 24 months from the date of manufacture. Handling: Prewash the beads three times with ice-cold PBS by brief low-speed centrifugation (500× g for 30 s) before use.
36. Rabbit IgG control antibody (Proteintech, catalog number: 30000-0-AP)
37. UFMylation Remnant Motif (K-ε-VG) kit (Micrometer Biotechnology, catalog number: WM302)
Note: Storage: Antibody-conjugated agarose beads at -20 °C; all other kit components at 4 °C. Shelf life: 12 months from the date of manufacture. Handling: Avoid repeated freeze-thaw cycles of the agarose beads; gently mix by inverting before use, do not vortex.
38. Color PAGE Gel Rapid Preparation kit (EpiZyme Biotechnology, catalog number: PG112)
39. Prestained protein ladder (EpiZyme Biotechnology, catalog number: WJ103)
40. Protease inhibitor cocktail (Roche, catalog number: 04693132001)
41. Anti-Myc affinity gel (Beyotime Biotechnology, catalog number: P2285)
42. Anti-DDRGK1 (Sigma-Aldrich, catalog number: HPA013373)
43. Anti-FLAG (Sigma-Aldrich, catalog number: F7425)
44. Anti-UFM1 (Abcam, catalog number: ab109305)
45. Anti-UFSP2 (Abcam, catalog number: ab185965)
46. Anti-Myc (Proteintech, catalog number: 60003-2-Ig)
47. Anti-HA (Cell Signaling Technology, catalog number: 3724)
48. Anti-GAPDH (ABclonal, catalog number: A19056)
49. Mouse IgG-agarose (Beyotime Biotechnology, catalog number: P2265)
50. Immobilon ECL Ultra Western HRP substrate (Millipore, catalog number: WBKLS0500)
51. Triethylammonium bicarbonate buffer (1 M) (Sigma-Aldrich, CAS number: 15715-58-9)
52. CaCl2 (Sigma-Aldrich, CAS number: 10043-52-4)
53. Trichostatin A (10 mM) (Selleck Chemicals, catalog number: S1045)
54. Nicotinamide (Sigma-Aldrich, CAS number: 98-92-0)
55. Urea (Sigma-Aldrich, CAS number: 57-13-6)
56. Sodium deoxycholate (Sigma-Aldrich, CAS number: 302-95-4)
57. 2-D Quant kit (Cytiva, catalog number: 80-6483-56)
Solutions
1. PBS (10×) (see Recipes)
2. PEI transfection reagent (see Recipes)
3. Lysis buffer (see Recipes)
4. SDS protein loading buffer (5×) (see Recipes)
5. Buffer A (see Recipes)
6. Denaturing lysis buffer (see Recipes)
7. NETN buffer (see Recipes)
8. SDS-PAGE running buffer (10×) (see Recipes)
9. Tris-glycine transfer buffer (10×) (see Recipes)
10. TBS (20×) (see Recipes)
11. 20% Tween-20 (see Recipes)
12. TBST (1×) (see Recipes)
13. Blocking buffer (see Recipes)
14. Antibody dilution buffer (see Recipes)
15. DMEM complete medium (see Recipes)
Recipes
1. PBS (10×)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Na2HPO4·12H2O | 100 mM | 36.28 g |
| NaCl | 1370 mM | 80 g |
| KCl | 27 mM | 2 g |
| KH2PO4 | 20 mM | 2.72 g |
| ddH2O | n/a | To 1,000 mL |
After the powder is completely dissolved, adjust the pH to 7.4, then vacuum-filter the solution for later use. Dilute the 10× PBS stock solution to a 1× working solution with ddH2O.
2. PEI transfection reagent
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| PEI | 1 mg/mL | 100 mg |
| ddH2O | n/a | To 100 mL |
Dissolve 100 mg of PEI powder in approximately 80 mL of ultrapure water with continuous stirring. Slowly add concentrated HCl to the PEI solution until pH < 2 and keep stirring until the powder is fully dissolved. Next, adjust the pH to 7.0 with NaOH solution, then top up with ultrapure water to a final volume of 100 mL. Mix well and filter the solution through a 0.22 μm sterile membrane filter in a biosafety cabinet. Prepare 1 mL aliquots and store at -20 °C. Avoid repeated freeze-thaw cycles.
3. Lysis buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Urea | 8 M | 48.05 g |
| Tris-HCl (1 M, pH 8.0) | 50 mM | 5 mL |
| NP-40 | 1% (v/v) | 1 mL |
| Sodium deoxycholate | 1% (m/v) | 1 g |
| DTT (1 M) | 5 mM | 500 μL |
| EDTA (0.5 M, pH 8.0) | 2 mM | 400 μL |
| Nicotinamide | 30 mM | 0.366 g |
| Trichostatin A (10 mM) | 3 μM | 30 μL |
| ddH2O | n/a | To 100 mL |
This lysis buffer is used for cell lysis in large-scale substrate identification by LC-MS/MS. Add 1× protease inhibitor cocktail before use.
4. SDS protein loading buffer (5×)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris-HCl (1 M, pH 6.8) | 250 mM | 25 mL |
| SDS | 10% (m/v) | 10 g |
| Glycerol | 50% (v/v) | 50 mL |
| DTT | 500 mM | 7.72 g |
| Bromophenol blue | 0.2% (m/v) | 0.2 g |
| ddH2O | n/a | To 100 mL |
After complete dissolution and thorough mixing, aliquot the solution at 1 mL per tube and store at -20 °C.
5. Buffer A
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris-HCl (1 M, pH 7.4) | 50 mM | 5 mL |
| NaCl (5 M) | 120 mM | 2.4 mL |
| NP-40 | 0.5% (v/v) | 0.5 mL |
| ddH2O | n/a | To 100 mL |
Store at 2–8 °C for up to 1 month.
6. Denaturing lysis buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris-HCl (1 M, pH 7.4) | 50 mM | 5 mL |
| EDTA (0.5 M, pH 8.0) | 0.5 mM | 100 μL |
| DTT (1 M) | 1 mM | 100 μL |
| 20% SDS | 2% (v/v) | 10 mL |
| ddH2O | n/a | To 100 mL |
Store at 2–8 °C for up to 1 month.
7. NETN buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris-HCl (1 M, pH 8.0) | 20 mM | 10 mL |
| EDTA (0.5 M, pH 8.0) | 1 mM | 1 mL |
| NaCl (5 M) | 100 mM | 10 mL |
| NP-40 | 0.5% (v/v) | 2.5 mL |
| ddH2O | n/a | To 500 mL |
Store at 2–8 °C for up to 1 month. The final pH of the prepared NETN buffer is 8.0 (adjust if necessary).
8. SDS-PAGE running buffer (10×)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 250 mM | 30.3 g |
| Glycine | 1920 mM | 144.1 g |
| SDS | 10 g/L | 10 g |
| ddH2O | n/a | To 1,000 mL |
Dilute the 10× running buffer to 1× running buffer with ddH2O.
9. Tris-glycine transfer buffer (10×)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tris base | 250 mM | 30.3 g |
| Glycine | 1920 mM | 144.1 g |
| ddH2O | n/a | To 1,000 mL |
Dilute the 10× transfer buffer to 1× transfer buffer with methanol and water to make a solution containing 25 mM Tris, 192 mM glycine, and 20% methanol.
10. TBS (20×)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| NaCl | 3000 mM | 175 g |
| Tris base | 500 mM | 60.6 g |
| KCl | 60 mM | 4.5 g |
| ddH2O | n/a | To 1,000 mL |
After complete dissolution, adjust the pH to 7.4 with dropwise diluted HCl under constant stirring, then bring the solution to a final volume of 1 L and store at room temperature.
11. 20% Tween-20
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Tween-20 | 20% | 10 mL |
| ddH2O | n/a | 40 mL |
| Total | n/a | 50 mL |
Store at 2–8 °C for up to 1 month.
12. TBST (1×)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| 20% Tween-20 | 0.1% | 5 mL |
| TBS (20×) | n/a | 50 mL |
| ddH2O | n/a | 940 mL |
13. Blocking buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Nonfat dry milk | 50 g/L | 5 g |
| 1× TBST | n/a | To 100 mL |
| ProClean 950 | 0.1% (v/v) | 100 μL |
| Total | n/a | 100 mL |
Store at 2–8 °C for up to 1 month.
14. Antibody dilution buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| BSA | 50 g/L | 5 g |
| TBST (1×) | n/a | To 100 mL |
| ProClean 950 | 0.1% (v/v) | 100 μL |
| Total | n/a | 100 mL |
Store at 2–8 °C for up to 1 month.
15. DMEM complete medium
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| DMEM with high glucose | 89% (v/v) | 445 mL |
| FBS | 10% (v/v) | 50 mL |
| Penicillin-streptomycin solution | 1% (v/v) | 5 mL |
| Total | n/a | 500 mL |
Store at 2–8 °C for up to 1 month.
Laboratory supplies
1. Immobilon®-PSQ PVDF membrane (Millipore, catalog number: ISEQ00010)
2. 100 mm cell culture dishes (Nunc, catalog number: 150466)
3. 1.5 mL microtubes (Axygen, catalog number: MCT-150-C)
4. 15 mL conical tube (KIRGEN, catalog number: KG2611)
5. 10 mL pipettes (Nunc, catalog number: 170356N)
6. 100–1,000 μL tips (KIRGEN, catalog number: KG1313)
7. 1–200 μL tips (KIRGEN, catalog number: KG1212)
8. 0.1–10 μL tips (KIRGEN, catalog number: KG1011)
9. Cell scraper (Corning, catalog number: 3010)
10. C18 SPE column (Phenomenex, catalog number: 8B-S100-AAK)
11. Vacuum concentrator (Thermo Fisher Scientific, catalog number: SPD111V-230)
12. C18 ZipTips (Merck Millipore, catalog number: ZTC18S096)
Equipment
1. Magnetic stirrer (IKA, model: C-MAG HS 7)
2. pH meter (Sartorius, model: PB-11)
3. Analytical balance (Mettler Toledo, model: MS105DU)
4. Biological safety cabinet (Thermo Scientific, model: 1386)
5. Pipette fillers (Thermo Scientific, catalog number: 9501)
6. Heating block (AOSHENG, model: K30)
7. 2–8 °C refrigerator (Thermo Scientific, model: PLR-386)
8. Biomedical freezer (Haier, model: DW-40L262)
9. Ultra-low temperature freezers (Thermo Scientific, model: 994)
10. Water-jacketed CO2 incubator (Thermo Scientific, model: 3111)
11. Vortex (Scientific Industries, model: G-560E)
12. Refrigerated centrifuge (Thermo Scientific, model: Sorvall ST 16R)
13. Centrifuge (Eppendorf, model: 5424)
14. Refrigerated centrifuge (Eppendorf, model: 5424R)
15. Electric water bath (Yuexin, model: HH-6)
16. Shaker (QILINBEIER, model: TS-8)
17. Ultrasonic cell disruptor (Scientz Biotechnology, model: SCIENTZ08 - IIIC)
18. Mixer (QILINBEIER, model: WH-986)
19. PowerPacTM Basic Power Supply (Bio-Rad Laboratories, catalog number: 1645050)
20. ChemiDoc MP Imaging System (Bio-Rad Laboratories, catalog number: 12003154)
Software and datasets
1. Image Lab (Bio-Rad Laboratories, Version 6.0, free to use)
2. MS proteomics data have been deposited to PRIDE (https://www.ebi.ac.uk/pride/archive/projects/PXD070408, PXD070408, 3/29/2026).
Procedure
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文章信息
稿件历史记录
提交日期: May 9, 2026
接收日期: Jun 12, 2026
在线发布日期: Jun 29, 2026
出版日期: Jul 20, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Liang, Q., Fang, Y., Dong, J., Yi, X. and Cong, Y. (2026). A Practical Experimental Protocol for Identification and Validation of UFMylation Substrate in Human Cells. Bio-protocol 16(14): e5758. DOI: 10.21769/BioProtoc.5758.
- Fang, Y., Wang, X., Dong, J., Yi, X., Lv, Z., Bu, C., Ma, X., Cong, Y. S. and Liang, Q. (2026). Optimization of protein UFMylation modification method and its application in substrate identification in human cells. J Biol Chem. 302(4): 111320. https://doi.org/10.1016/j.jbc.2026.111320
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分类
生物化学 > 蛋白质 > 翻译后修饰
分子生物学 > 蛋白质 > 泛素化
细胞生物学 > 细胞工程 > CRISPR-cas9
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