Protocol for proteomic profiling of hypusinated protein candidates using a clickable spermidine probe

Summary

Hypusination is a unique posttranslational modification in which deoxyhypusine synthase (DHPS) transfers an aminobutyl moiety from spermidine to specific lysine residues, followed by deoxyhypusine hydroxylase (DOHH)-mediated hydroxylation. Here, we present a protocol that enables proteome-wide identification of candidate hypusinated proteins. We describe steps for the synthesis of a clickable alkynyl-spermidine probe, DHPS-dependent metabolic labeling in cells, click chemistry-mediated biotinylation, streptavidin-based enrichment, and subsequent mass spectrometry analysis of probe-labeled proteins. We also describe the procedures for data processing and statistical analysis.

For complete details on the use and execution of this protocol, please refer to Zhang et al.¹

Graphical abstract

Highlights

• •Instructions for synthesizing a clickable spermidine probe
• •Protocol for metabolic labeling of cultured cells with the clickable spermidine probe
• •Click chemistry procedure for biotinylation of probe-labeled proteins
• •Workflow for identifying hypusinated protein candidates by mass spectrometry

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Hypusination is a unique posttranslational modification in which deoxyhypusine synthase (DHPS) transfers an aminobutyl moiety from spermidine to specific lysine residues, followed by deoxyhypusine hydroxylase (DOHH)-mediated hydroxylation. Here, we present a protocol that enables proteome-wide identification of candidate hypusinated proteins. We describe steps for the synthesis of a clickable alkynyl-spermidine probe, DHPS-dependent metabolic labeling in cells, click chemistry-mediated biotinylation, streptavidin-based enrichment, and subsequent mass spectrometry analysis of probe-labeled proteins. We also describe the procedures for data processing and statistical analysis.

Before you begin

Protein hypusination is a unique post-translational modification in which spermidine donates an aminobutyl moiety to a specific lysine residue in target proteins mediated sequentially by deoxyhypusine synthase (DHPS) and then hydroxylated by deoxyhypusine hydroxylase (DOHH).^2,3 This hypusination modification has long been considered restricted to a single protein substrate eIF5A, identified in the 1980s by two-dimensional electrophoresis.⁴ This protocol describes the synthesis and use of a clickable alkyne-functionalized spermidine probe to enable DHPS-dependent metabolic labeling and enrichment of probe-labeled proteins for proteomic analysis which was used to identify 1627 candidate hypusinated protein targets including RIPK1.¹

Prior to starting the protocol, the alkyne-functionalized spermidine probe should be designed, synthesized and characterized. The synthesis of the probe is accomplished through sequential alkylation reactions followed by global deprotection step to yield the probe as its tris(trifluoroacetate) salt. The purity and identity of the intermediate and final product should be confirmed by NMR and, if possible, mass spectrometry. The probe should be dissolved in water or appropriate buffer and stored at −20°C in aliquots to avoid repeated freeze–thaw cycles.

Mouse embryonic fibroblasts (MEFs), including wild-type and DHPS knockout cells, should be maintained under standard culture conditions and confirmed to be free of mycoplasma contamination. The use of DHPS-deficient cells provides an essential control to assess labeling specificity of spermidine mediated hypusination. For optimal labeling efficiency, cells should be in a healthy proliferative state prior to treatment. The polyamine oxidase inhibitor MDL-72527 is used to stabilize the intracellular spermidine probe.

The click probe should be added to cells maintained in normal culture medium. The excess free click probe needs to be removed before click chemistry reaction. Efficient click chemistry and enrichment depend on protein quality and buffer compatibility. Cell lysis should be performed in SDS-containing buffer supplemented with protease inhibitors (cOmplete EDTA-free protease inhibitor cocktail) and N-Ethylmaleimide (NEM) to block serine/cysteine proteases and prevent post-lysis protein degradation, thereby ensuring efficient solubilization and stabilization of proteins. Copper-catalyzed azide–alkyne cycloaddition (CuAAC) reagents, including azide-biotin, CuSO₄, TCEP, and TBTA ligand, should be in good quality and freshly prepared to maintain reaction efficiency. Streptavidin beads should be equilibrated prior to use, and extensive washing is critical to reduce nonspecific binding.

For downstream mass spectrometry analysis, the users should ensure access to a high-resolution LC–MS/MS system and appropriate data analysis pipelines. Protein samples should be of sufficient quantity and quality, and experimental design should include biological replicates and appropriate controls to support confident identification of probe-labeled proteins.

The hit selection protocol is important for the identification of high-confidence candidate proteins. Parallel controls and DHPS knockout are used to exclude any non-specific hits whose enrichment is not reduced by excess spermidine or by DHPS knockout. Potential non-specific hits such as azide-alkyne-thiol adduct formation during the bioorthogonal ligation reaction can be eliminated from final analysis by the lack of DHPS-dependency or inability to be competed away by excess spermidine. Any such non-specific products should be excluded from the final hit list.

Innovation

Historically, protein hypusination was believed to be restricted to the translation initiation factor eIF5A. This protocol introduces a transformative chemical proteomic workflow that overcomes previous technical limitations, enabling the first systematic, proteome-wide identification of hypusination candidate proteins.¹ The workflow utilizes a clickable alkynyl-spermidine probe coupled with copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated biotinylation and streptavidin-based enrichment, allowing for the capture and proteomic profiling of rare, low-abundance hypusinated proteins otherwise undetectable by conventional mass spectrometry. It ensures rigorous biological specificity by integrating DHPS-knockout (KO) cell lines as negative controls and utilizing the polyamine oxidase inhibitor MDL-72527 to maintain probe stability. By merging synthetic chemistry with high-resolution LC-MS/MS, this method provides a robust platform for discovering new hypusinated protein candidates.

Preparation of the clickable alkynyl-spermidine probe

Timing: 7 days

A three-step synthetic route (Figure 1) is designed to obtain the compound N¹-(3-ammoniopropyl)-N⁴-(prop-2-yn-1-yl)butane-1,4-diaminium tris(2,2,2-trifluoroacetate) (compound 1). In this route, we sequentially convert the readily available precursor (compound 2) into two intermediates (compound 3 and 4) and remove the Boc protecting groups from compound 4 with trifluoroacetic acid (TFA) to afford the desired clickable probe as its tris(trifluoroacetate) salt (compound 1). The steps below describe the detailed procedures for each stage of the synthesis.

Note: All reagents were purchased from commercial suppliers and used without further purification.

• 1.
  First step of synthesis of tert-butyl(4-bromobutyl) (prop-2-yn-1-yl)carbamate (compound 3). Please refer to Figure 1 and Table 1.

  • a.
    Weigh compound 2 (3.10 g) in a 100 mL round-bottom flask containing a magnetic stirring bar.
  • b.
    Add anhydrous N,N-dimethylformamide (DMF, 50 mL) to the flask using a syringe and cool the flask in an ice bath for 10 min.
  • c.
    Weigh sodium hydride (0.96 g) and add it slowly to the solution. After addition, stir the mixture in an ice bath for 30 min.
  • d.
    Weigh 1,4-dibromobutane (5.17 g) and add it to the reaction mixture via syringe.
  • e.
    Heat the reaction to 50 °C and stir for 16 h at this temperature.
  • f.
    Treat the reaction mixture with saturated aqueous NH₄Cl solution (10 mL) and extract once with ethyl acetate (EtOAc) (25 mL).
  • g.
    Wash the organic layer with saturated aqueous NH₄Cl solution (10 mL × 3) followed by brine (10 mL × 2).
  • h.
    Dry the organic layer over anhydrous Na₂SO₄.
  • i.
    Filter the mixture through a Büchner funnel.
  • j.
    Concentrate the filtrate under reduced pressure.
  • k.
    Purify the residue by flash silica gel chromatography (petroleum ether as eluent) to afford compound 3 (4.17 g, 72% yield).
  • l.
    Characterize the title product by ¹H NMR spectroscopy (Figure 2A). ¹H NMR (400 MHz, DMSO-d₆) δ 3.97 (s, 2H), 3.56-3.52 (t, J = 8.0 Hz, 2H), 3.26-3.23 (t, J = 8.0 Hz, 2H), 3.17-3.15 (t, J = 4.0 Hz, 1H), 1.81-1.74 (p, J = 8.0 Hz, 2H), 1.67 (m, 2H), 1.41 (s, 9H).
  • m.
    Characterize the title product by ¹³C NMR spectroscopy (Figure 2B). ¹³C NMR (101 MHz, DMSO-d₆) δ 154.7, 80.9, 79.8, 74.3, 45.8, 35.2, 30.0, 28.4, 26.6.

Pause point: The purified compound 3 can be stored in a sealed flask under nitrogen at −20 °C for several days.

• 2.
  Second step of synthesis of tert-butyl(4-((3-((tert-butoxy carbonyl)amino)propyl)amino) butyl) (prop-2-yn-1-yl)carbamate (compound 4). Please refer to Figure 1 and Table 2.

  • a.
    Weigh compound 3 (1.00 g) in a 100 mL round-bottom flask containing a magnetic stirring bar.
  • b.
    Add acetonitrile (20 mL) to the flask using a syringe.
  • c.
    Weigh potassium carbonate (2.66 g) and add it to the solution.
  • d.
    Weigh tert-butyl (3-aminopropyl) carbamate (0.57 g) and add it to the mixture.
  • e.
    Heat the reaction to 50 °C and maintain at this temperature for 15 h.
  • f.
    Treat the reaction mixture with saturated aqueous NH₄Cl solution (10 mL) and extract once with EtOAc (25 mL).
  • g.
    Wash the organic layer with saturated aqueous NH₄Cl solution (10 mL × 3) followed by brine (10 mL × 2)
  • h.
    Dry the organic layer over anhydrous Na₂SO₄.
  • i.
    Filter the mixture through a Büchner funnel.
  • j.
    Concentrate the filtrate under reduced pressure.
  • k.
    Purify the resulting residue by flash silica gel chromatography using a gradient of 0–5% MeOH in CH₂Cl₂ to afford compound 4 (405.00 mg, 31% yield).
  • l.
    Characterize the title product by ¹H NMR spectroscopy (Figure 3A). ¹H NMR (400 MHz, DMSO-d₆) δ 6.96 (m, 1H), 3.99 (s, 2H), 3.25-3.22 (m, 2H), 3.19-3.18 (m, 1H) 3.02-2.97 (q, J = 8.0 Hz, 2H), 2.90-2.83 (m, 4H), 1.74-1.67 (p, J = 8.0 Hz, 2H), 1.55 (m, 4H), 1.41 (s, 9H), 1.39 (s, 9H), 1.26-1.24 (m, 1H).
  • m.
    Characterize the title product by ¹³C NMR spectroscopy (Figure 3B). ¹³C NMR (101 MHz, DMSO-d₆) δ 156.2, 154.7, 80.9, 79.8, 78.3, 47.1, 46.0, 45.2, 37.5, 28.7, 28.46, 26.8, 23.4.

Pause point: The purified compound 4 can be stored in a sealed flask under nitrogen at −20 °C for several days.

• 3.
  Third step of synthesis of N¹-(3-ammoniopropyl)-N⁴-(prop-2-yn-1-yl) butane-1,4-diaminium tris(2,2,2-trifluoroacetate) (compound 1). Please refer to Figure 1 and Table 3.

  • a.
    Weigh compound 4 (405.00 mg) in a 100 mL round-bottom flask containing a magnetic stirring bar.
  • b.
    Add CH₂Cl₂ (10 mL) to the flask using a syringe.
  • c.
    Cool the flask in an ice bath.
  • d.
    Add CF₃CO₂H (TFA, 2.0 mL) using a syringe.
  • e.
    Stir the reaction in an ice bath for 1 h, and then concentrate to dryness.
  • f.
    Triturate the residue with toluene (5.0 mL) for 30 min.
  • g.
    Collect the solid by filtration and air-dry to afford compound 1 as the tris(trifluoroacetate) salt (470.00 mg, 84% yield).
  • h.
    Characterize the title product by ¹H NMR spectroscopy (Figure 4A). ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (br, 2H), 8.68 (br, 2H), 7.88 (br, 3H),3.93-3.92 (d, J = 4.0 Hz, 2H), 3.75 (m, 1H), 2.98-2.85 (m, 8H), 1.91-1.83 (p, J = 8.0 Hz 2H), 1.62 (m, 4H).
  • i.
    Characterize the title product by ¹³C NMR spectroscopy (Figure 4B). ¹³C NMR (101 MHz, DMSO-d₆) δ 159.1-158.8 (d, J = 31.3 Hz), 117.6 (d, J = 299.0 Hz), 79.9, 75.5, 46.6, 45.8, 44.3, 36.6, 36.1, 24.2, 23.1, 23.1.
  • j.
    Characterize the title product by high resolution mass spectrometry (Figure 5). A prominent peak is expected at m/z 184.18083, which aligns with the calculated theoretical value m/z 184.18137 (mass error < 10 ppm)

Pause point: The purified compound 1 as its tris(trifluoroacetate) salt can be stored in a sealed flask under nitrogen at −20 °C for several months.

Figure 1.

Three-step synthesis of the clickable alkynyl-spermidine probe (compound 1)

(i) 1,4-dibromobutane, NaH, DMF, 50 ^oC, 16 h; (ii) tert-butyl (3-aminopropyl) carbamate, K₂CO₃, CH₃CN, 50 ^oC, 15h; (iii) CF₃CO₂H, CH₂Cl₂, 0°C, 1 h.

Table 1.

Reagents for synthesis of compound 3

Reagent	Formula weight	Equivalent	Amount
Compound 2 (N-Boc-2-propyn-1-amine)	155.20 Da	1.00	3.10 g
Sodium hydride (60% in mineral oil)	24.00 Da	1.20	0.96 g
1,4-dibromobutane	215.91 Da	1.20	5.17 g

Figure 2.

Characterization of compound 3 by NMR spectroscopy

(A and B) Characterization of compound 3 by ¹H NMR spectroscopy (A) and ¹³C NMR spectroscopy (B).

Table 2.

Reagents for synthesis of compound 4

Reagent	Formula weight	Equivalent	Amount
Compound 3	290.20 Da	1.00	1.00 g
Potassium carbonate	138.20 Da	5.59	2.66 g
tert-butyl (3-aminopropyl) carbamate	174.24 Da	0.95	0.57 g

Figure 3.

Characterization of compound 4 by NMR spectroscopy

(A and B) Characterization of compound 4 by ¹H NMR spectroscopy (A) and ¹³C NMR spectroscopy (B).

Table 3.

Reagents for synthesis of compound 1

Reagent	Formula weight	Equivalent	Amount
Compound 4	383.53 Da	1.00	405.00 mg
CF3CO2H	114.02 Da	excess	2.00 mL

Figure 4.

Characterization of compound 1 by NMR spectroscopy

(A and B) Characterization of compound 1 by ¹H NMR spectroscopy (A) and ¹³C NMR spectroscopy (B).

Figure 5.

Characterization of compound 1 by Orbitrap high-resolution mass spectrometry

Preparation of the DHPS knockout MEFs

Timing: 7 days

• 4.
  Plasmid construction for knockout of Dhps in cells.

  • a.
    Annealing: Phosphorylate and anneal sgDhps (or sgGFP) forward and reverse oligos. Prepare the annealing reaction mixtures for sgDhps (experimental group) and sgGfp (control group) in separate reaction tubes. Each 10 μL reaction system should contain 1 μL of the corresponding forward oligo and 1 μL of the reverse oligo.
  • b.
    Digestion: Digest lentiCRISPR v2 with BsmBI, resolve the mixture on a 0.8% (w/v) agarose gel.
  • c.
    Isolate the expected ∼13 kb linearized backbone band.
  • d.
    Purify the backbone using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s protocol (https://www.qiagen.com/en-US/resources/download/Protocols/hb-0901-003-1114358-pcard-qq-pcr-gel-cleanup-kit-0718-ww).
  • e.
    Ligation: Ligate the annealed oligos into the digested backbone.
  • f.
    Transformation: Transform the ligation product into Stbl3 competent cells via heat-shock at 42 °C for 45 s, followed by incubation on ice for 2 min.
  • g.
    Recover the cells in 500 μL of SOC medium at 37 °C for 1 h with shaking at 225 rpm.
  • h.
    Plate the cells onto LB agar containing 100 μg/mL ampicillin.
  • i.
    Confirm correct sgRNA insertion by Sanger sequencing of individual colonies using a U6 forward primer, provided by a commercial sequencing service.
  • j.
    Extract endotoxin-free plasmids from sequence-verified clones.

Annealing reaction master mix

Reagent	Amount
sgDhps forward oligo (5′-CACCGGCGCATGGTAATCTACGCCG-3′) (100 μM)	1 μL
sgDhps reverse oligo (5′-AAACCGGCGTAGATTACCATGCGCC-3′) (100 μM)	1 μL
sgGfp forward oligo (5′-CACCGGGCGAGGAGCTGTTCACCG-3′) (100 μM)	1 μL
sgGfp reverse oligo (5′-AAACCGGTGAACAGCTCCTCGCCC-3′) (100 μM)	1 μL
10× T4 Polynucleotide Kinase (PNK) reaction buffer	1 μL
T4 PNK (Polynucleotide Kinase)	0.5 μL
ddH2O	6.5 μL
Total volume	10 μL

Annealing conditions

Temperature	Time
37 °C	30 min
95 °C	5 min
Ramp down to 25 °C at a rate of 0.1 °C per second (or −5 °C every 1 min)

Digestion master mix

Reagent	Amount
lentiCRISPR v2 Plasmid	5 μg
BsmBI-v2	1 μL
10x rCutSmart buffer/NEBuffer	5 μL
ddH2O	Up to 50 μL

Digestion conditions

Temperature	Time
55 °C	4 h

Ligation master mix

Reagent	Amount
Digested lentiCRISPR v2	50 ng
Diluted Annealed Oligos (1:100)	1 μL
10x T4 ligation buffer	1 μL
T4 DNA ligase	0.5 μL
ddH2O	Up to 10 μL

Ligation conditions

Temperature	Time
16 °C	12∼15 h

CRITICAL: Sanger sequencing using the U6 forward primer is required to ensure that the sgRNA was inserted correctly and without mutations in the scaffold.

Pause point: Endotoxin-free plasmids can be stored at 4°C for several months.

• 5.
  Lentivirus production (HEK293T).

  • a.
    Transfection: Culture HEK293T cells in a 6-well plate until they reach 70–80% confluence.
  • b.
    Prepare a transfection mixture containing 0.9 μg of lentiCRISPR-sgDhps or lentiCRISPR-sgGfp, 0.7 μg of psPAX2, and 0.4 μg of pMD2.G (4:3:2 mass ratio; 2 μg total DNA).
  • c.
    Mix the plasmids with linear polyethylenimine.
  • d.
    Add the mixture to a single well containing 2 mL of culture medium.
  • e.
    Medium Change: Replace the medium with fresh DMEM supplemented with 10% FBS after 6–8 h.
  • f.
    Harvest: Collect viral supernatants at 48 h and 72 h post-transfection.
  • g.
    Filter the viral supernatants through a 0.45 μm nitrocellulose membrane.

Pause point: Filtered viral supernatants can be stored at −80 °C for several months or at 4 °C for several days, though a slight loss in virus titer may occur with freeze-thaw cycles.

• 6.
  MEFs transduction and selection.

  • a.
    Infection: Infect low-passage MEFs (at 50% confluence in 2 mL of culture medium) with 1 mL of viral supernatant in the presence of Polybrene (8 μg/mL).
  • b.
    Recovery: Replace with fresh medium 24 h post-infection.
  • c.
    Selection: Add puromycin (3 μg/mL) at 48 h post-infection.
  • d.
    Maintenance: Continue selection for 3–5 days until non-infected control cells are completely eliminated.

CRITICAL: Always include a non-infected control well when adding puromycin. Selection is only successful once 100% of the control cells have died.

• 7.
  Validation of knockout efficiency.

  • a.
    Lyse the cells using RIPA lysis and extraction buffer and perform western blot analysis.
  • b.
    Detect DHPS protein levels using a DHPS-specific antibody to confirm knockout efficiency.

CRITICAL: Western blot validation of DHPS knockout is mandatory before proceeding to click chemistry reaction to ensure the biological model is valid.

Pause point: DHPS knockout MEFs can be stored in liquid nitrogen for several years.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Streptavidin-HRP	Beyotime	Cat#A0305
DHS antibody (A-10)	Santa Cruz Biotechnology	Cat#sc-365077
Chemicals, peptides, and recombinant proteins
Compound 2 (N-Boc-2-propyn-1-amine)	Accela ChemBio	Cat#SY025866
Sodium hydride (60% in mineral oil)	Macklin	Cat#S817935-100g
1,4-dibromobutane	Macklin	Cat#D665801-5g
Potassium carbonate	Macklin	Cat#P816302-500g
tert-Butyl (3-aminopropyl) carbamate	Macklin	Cat#N803413-5g
CF3CO2H	Macklin	Cat#T818781-100ml
Urea	MERCK	Cat#U5128
BsmBI-v2 restriction enzyme	New England Biolabs	Cat#R0739S
10× T4 DNA ligase buffer	New England Biolabs	Cat#B0202S
10× T4 Polynucleotide Kinase (PNK) reaction buffer	New England Biolabs	Cat#B0201S
T4 Polynucleotide Kinase	New England Biolabs	Cat#M0201S
10× rCutSmart buffer/NEBuffer	New England Biolabs	Cat#B6004S
T4 DNA ligase	New England Biolabs	Cat#M0202S
Agarose	Bio-Rad	Cat#1613101
QIAquick Gel extraction kit	Qiagen	Cat#28704
Pre-poured LB agar plates with 100 μg/mL ampicillin	Teknova	Cat#L1110
Endotoxin-free plasmid extraction kit	Qiagen	Cat#12943
Polyethylenimine	Polysciences	Cat#23966-1
Methanol, LC-MS grade	MERCK	Cat#34966
Stbl3 competent cells	Thermo Fisher Scientific	Cat#C737303
SOC medium	Thermo Fisher Scientific	Cat#15544034
Tris(2-carboxyethyl)phosphine (TCEP)	MERCK	Cat#C4706
Iodoacetamide (IAA)	MERCK	Cat#I1149
CaCl2	MERCK	Cat#223506
Tris base	MERCK	Cat#T1503
Trypsin	Promega	Cat#V511C
C18 desalting spin columns	Thermo Fisher Scientific	Cat#87784
C18 resin (1.9 μm)	Dr. Maisch GmbH	Cat#r119.aq.0003
Formic acid, LC-MS grade	Thermo Fisher Scientific	Cat#85178
Acetonitrile, LC-MS grade	Fisher Chemical	Cat#A955
Water, LC-MS grade	Fisher Chemical	Cat#W6
Hydrochloric acid; analytical grade	Sinopharm	Cat#10011018
TBTA	Sigma	Cat#678937
CuSO4	Sigma	Cat#C1297
Biotin-azide	Cayman	Cat#13040
MDL72527	Sigma	Cat# M2949
Spermidine	Sigma	Cat#S2501
RIPA lysis and extraction buffer	Thermo Fisher Scientific	Cat#89900
cOmplete EDTA-free protease inhibitor cocktail	Roche	Cat#C762Q77
N-Ethylmaleimide	Sigma	Cat#34115-M
SDS-PAGE loading buffer (reduced)	Bioswamp	Cat#WB9207
SDS	Sigma	Cat#436143
Pierce ECL western blotting substrate	Thermo Fisher Scientific	Cat# 32209
Non-fat dry milk	Bio-Rad	Cat #1706404XTU
10× Tris/Glycine/SDS running buffer	Bio-Rad	Cat #1610772
4–15% criterion TGX stain-free protein gels	Bio-Rad	Cat#5678083
TBST	Thermo Fisher Scientific	Cat#28360
Transfer buffer	Thermo Fisher Scientific	Cat#J63664.K2
BSA	CST	Cat#9998
DMEM	Thermo Fisher Scientific	Cat#C11995500
FBS	Thermo Fisher Scientific	Cat#10099141
Penicillin-Streptomycin	Thermo Fisher Scientific	Cat#15140122
PBS	Thermo Fisher Scientific	Cat#10010023
Pierce™ high capacity streptavidin agarose	Thermo Fisher Scientific	Cat#20361
Pierce™ BCA Protein Assay Kit	Thermo Fisher Scientific	Cat#23227
PageRuler™ Prestained Protein Ladder, 10 to 180 kDa	Thermo Fisher Scientific	Cat#26616
Experimental models: Cell lines
MEFs	ATCC	CRL-2991
DHPS knockout MEFs	N/A	N/A
Software and algorithms
MaxQuant	Max Planck Institute for Biochemistry	Version: 2.0.3.0
Perseus	Max Planck Institute for Biochemistry	Version:2.0.7.0
Excel	Microsoft Office	Version: office 2019
Other
Thermo Scientific Nunc EasYDish 150 mm dish	Thermo Fisher Scientific	Cat#150462
Criterion Vertical Electrophoresis Cell	Bio-Rad	Cat#1656001
Trans-Blot SD Semi-Dry Transfer Cell	Bio-Rad	Cat#1703940
Digital Dry Bath/Block Heater	Thermo Fisher Scientific	Cat#88870010
Nitrocellulose membrane	Bio-Rad	Cat#1620112
X-Ray Film	Thomas Scientific	Cat#1148B77
EASY-nLC™ 1200 system	Thermo Fisher Scientific	Cat#LC140
Trap Column	Thermo Fisher Scientific	Cat#164946
Q-Exactive HFX mass spectrometer	Thermo Fisher Scientific	Cat#0726042

Materials and equipment

8 M urea buffer (prepare fresh)

Reagent	Final concentration	Amount
Urea	8 M	0.48 g
500 mM Tris-HCl pH 8.5	100 mM	0.2 mL
ddH2O	N/A	0.44 mL
Total	N/A	1 mL

Digestion buffer components (prepare fresh)

Reagent	Concentration (stock)	Dilution factor
TCEP	500 mM	100×
IAA	500 mM	50×
CaCl2	100 mM	100×
Tris-HCl pH 8.5	100 mM	1×
Trypsin	0.5 μg/μL	N/A

Note: For maximal reagent stability and activity, it is recommended to prepare fresh 500 mM TCEP and 500 mM IAA solutions from solid reagents immediately before use. Alternatively, 500 mM stock solutions may be prepared, aliquoted, and stored at −20 °C for up to one year. In this case, aliquots should be thawed and diluted immediately before use.

Step-by-step method details

Click chemistry reaction and streptavidin pull-down

Timing: 2 days

• 1.Pre-treat wild-type (WT) or DHPS knockout (KO) mouse embryonic fibroblasts (MEFs) in 150 mm dishes (at 70–80% confluence in 20 mL of culture medium) with 20 μM MDL-72527 (a polyamine oxidase inhibitor) for 1 h in DMEM supplemented with 10% FBS.
• 2.Incubate the cells in the same culture medium as in step 1 with either 400 μM alk-spermidine probe or spermidine for 4 h, or in the case of competition experiments, co-treat cells with 400 μM alk-spermidine and 2 mM spermidine for 4 h.
• 3.Wash the cells twice with 12 mL of 1× PBS buffer at 22–28 °C.
• 4.Harvest the cells and lyse each dish in 500 μL of 1× PBS buffer containing 1% SDS, 10 mM N-Ethylmaleimide (NEM), and 1× protease inhibitor cocktail (diluted from a 100× stock in DMSO).
• 5.Perform sonication in an ice bath at 20% amplitude for 5 cycles (10 s on, 20 s off per cycle) to complete cell lysis.
• 6.Clarify lysates by centrifugation at 14,000 × g for 10 min at 4 °C.
• 7.Determine the protein concentration in the supernatant obtained in step 6 using a BCA assay kit.
• 8.Aliquot 1 mg of protein (∼1 mg/mL) and perform CuAAC click chemistry by adding the reagents in the following order: (1) azide-biotin (10 mM stock), (2) TBTA (20 mM stock), (3) CuSO₄ (50 mM stock), and (4) freshly prepared TCEP (50 mM stock) to final concentrations of 200 μM, 400 μM, 1 mM, and 1 mM, respectively.
• 9.After 30 min of reaction, add an equal volume of 50 mM TCEP (as used in step 8) to reach a final theoretical concentration of 2 mM to maintain reaction activity.
• 10.Continue the incubation in step 9 at 22–28 °C for a total of 1 h.
• 11.Precipitate the proteins by adding 4 mL of ice-cold methanol to the 1 mL reaction mixture, and incubate at −20 °C for 1∼3 h.
• 12.Centrifuge the mixture in step 11 at 4 °C at 14,000 × g for 20 min to pellet the proteins.
• 13.Re-suspend the protein pellets in 500 μL of 1% SDS using sonication.
• 14.Dilute the SDS concentration to 0.2% with PBS.
• 15.Incubate the protein mixture with 40 μL streptavidin agarose beads for 2 h with constant end-over-end rotation at 22–28 °C.
• 16.Wash the beads with 1% SDS (6 × 1 mL) for 10 min each time, carefully removing the supernatant after each wash.
• 17.Wash next with 0.1% SDS (6 × 1 mL) for 10 min each time, ensuring complete removal of the supernatant between steps.
• 18.Perform a final wash sequence with PBS (6 × 1 mL) for 10 min each time, ensuring complete removal of the supernatant between steps.
• 19.Elute the enriched proteins in 80 μL 1× loading buffer at 95 °C for 10 min to prepare for western blot analysis.

CRITICAL: The stock solutions of TCEP and CuSO₄ should be freshly prepared. The solution should be mixed well to ensure the formation of the active Cu(I) catalyst.

CRITICAL: Pretreatment with MDL-72527 is required to maintain probe stability. Polyamine oxidase can promote the degradation of spermidine click probe to reduce the efficiency of probe labeling. In addition, polyamine oxidation generates reactive aldehydes and ROS, which may further compromise labeling specificity and introduce artifactual modifications.^5,6,7 Therefore, inhibition of polyamine oxidation (e.g., with MDL72527) is essential to preserve probe integrity and enable accurate identification of click probe-labeled targets, especially for low abundant protein targets.

Note: The addition of NEM in lysis buffer is used to block free cysteines and help minimize thiol-associated nonspecific probe labeling. NEM blocks free cysteines by undergoing a Michael addition at near-neutral pH, forming an irreversible covalent thioether bond with the sulfhydryl group (-SH) to permanently mask it from subsequent labeling or oxidation.

Note: In step 1, the MDL-72527 is added from a 100 mM stock solution dissolved in DMSO, keeping the final DMSO concentration at 0.02% (v/v) to eliminate potential vehicle toxicity.

Note: Both reagents in step 2 are added from 1200 mM stock solutions, keeping the final DMSO concentration at or below 0.20% (v/v) to avoid cytotoxicity. Due to the hygroscopic and unstable properties of spermidine, the stock solutions must be freshly prepared in anhydrous DMSO immediately before each use.

Note: Complete removal of the excess, unreacted clickable spermidine probe (as described in step 3) is essential prior to the click chemistry reaction in step 8 to prevent high background signal. If necessary, a protein precipitation step may also be performed prior to step 8.

Note: For mass spectrometry analysis, proceed only up to step 18 without performing the elution step.

Pause point: Boiled western blot samples can be stored at 4 °C for up to two days.

Western blot analysis of probe-labeled proteins enriched by streptavidin agarose after click chemistry

Timing: ∼8 h

• 20.Load 20 μL of samples prepared in step 19 into the 4–15% criterion TGX stain-free SDS-PAGE gel.
• 21.Fill the electrophoresis tank with 1× Tris-Glycine-SDS running buffer
• 22.Run the gel at 80 V for the stacking phase, then increase to 120 V for the resolving phase until the tracking dye reaches the bottom.
• 23.Assemble the transfer sandwich.
• 24.Transfer the proteins from the gel to the nitrocellulose membrane in transfer buffer at a constant current of 250 mA for 90 min under chilled conditions.
• 25.Block the membrane in 5% non-fat milk in TBST for 1 h at 22–28 °C.
• 26.Wash the membrane three times for 5 min each with TBST.
• 27.Dilute streptavidin-HRP at 1:2000 in 5% BSA/TBST.
• 28.Incubate the membrane with the diluted solution at 22–28 °C for 2 h with gentle agitation.
• 29.Wash the membrane with TBST (3 × 10 min) to remove unbound streptavidin-HRP.
• 30.Incubate the membrane with ECL chemiluminescence substrate for 1 min.
• 31.Expose the membrane to X-ray film to capture the chemiluminescent signal.

On-bead digestion of biotin pull-down samples and desalting

Timing: ∼16 h

This section describes the preparation of peptide samples of enriched probe-labeled proteins using the on-bead digestion method for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

• 32.Following step 18, remove as much of the remaining liquid as possible.
• 33.Resuspend the streptavidin agarose beads in 50 μL of 8 M urea solution.
• 34.Add 0.5 μL of 0.5 M TCEP to the mixture at a final concentration of 5 mM.
• 35.Incubate the mixture at 22–28 °C for 20 min.
• 36.Add 1 μL of 0.5 M IAA to the mixture at a final concentration of 10 mM.
• 37.Incubate the mixture at 22–28 °C in the dark for 15 min.
• 38.Add 150 μL of 100 mM Tris-HCl (pH 8.5, prepare with Tris base and adjust pH with hydrochloric acid) buffer to dilute the urea concentration to 2 M.
• 39.Add 2 μL of 100 mM CaCl₂ solution to the mixture at a final concentration of 1 mM.
• 40.Add 2 μL of trypsin (0.5 μg/μL, supplied as a frozen solution in 50 mM acetic acid) to the mixture.
• 41.Incubate the mixture at 37 °C with shaking at 800 rpm for 12 h.
• 42.Add 10 μL of 100% formic acid (FA) to the mixture to terminate the digestion.
• 43.Centrifuge the enzymatically digested sample at 20,000 × g and 4 °C for 15 min.
• 44.Transfer the resulting supernatant to a new eppendorf (EP) microcentrifuge tube for desalting.
• 45.
  Insert the C18 desalting spin columns into the cap of a 1.5 mL EP tube.

  • a.
    Add 200 μL of acetonitrile to the column.
  • b.
    Centrifuge the EP tube in a microcentrifuge (2,000 × g, 22–28 °C) to activate the C18 packing material.
  • c.
    Discard the flow-through.
• 46.Load the C18 desalting columns with 200 μL of 0.1% formic acid (FA) solution and centrifuge it in a microcentrifuge (2,000 × g, 22–28 °C) to equilibrate the C18 desalting columns.
• 47.Discard the flow-through, and repeat step 46 twice.
• 48.Load the supernatant sample obtained in step 44 onto the C18 desalting columns and centrifuge it in a microcentrifuge (2,000 × g, 22–28 °C).
• 49.Repeat step 48 twice.
• 50.Discard the final flow-through.
• 51.Load the C18 desalting columns with 200 μL of 0.1% FA solution and centrifuge it in a microcentrifuge (2,000 × g, 22–28 °C), and discard the flow-through.
• 52.Repeat step 51 twice.
• 53.
  Transfer the C18 desalting columns to a new 1.5 mL EP tube.

  • a.
    Add 100 μL of peptide elution buffer (containing 70% acetonitrile and 0.1% FA).
  • b.
    Centrifuge the EP tube in a microcentrifuge (2,000 × g, 22–28 °C), and collect the eluate into a new 1.5 mL EP tube.
  • c.
    Repeat the elution step a and b twice and pool all eluates.
• 54.Dry the pooled peptide eluate under vacuum at 4 °C.

Note: Before the experiment, prepare a fresh 8 M urea (in 100 mM Tris-HCl, pH 8.5) solution.

Note: For each step involving solvent replacement or sample loading, the remaining liquid from the previous step should be centrifuged until the liquid level aligns with the top of the C18 packing material.

Pause point: The dried sample can be stably stored at −20 °C for several months to one year until further analysis by LC-MS/MS.

LC-MS/MS data acquisition

Timing: ∼1.5 days

Peptides processed in previous steps are now analyzed by LC-MS/MS.

• 55.Dissolve the dried peptide sample in 15 μL of freshly prepared 0.1% FA solution and vortex the mixture.
• 56.Centrifuge the mixture at 20,000 × g and 4 °C for 15 min.
• 57.Subsequently, transfer 10 μL of the supernatant to an autosampler vial for LC-MS/MS analysis.
• 58.Load peptide samples by injecting 2 μL onto a trap column (a commercial column, 70 mm × 75 μm i.d., packed with 3 μm C18 resin).
• 59.
  Separate them on a C18 analytical column (150 mm × 100 μm i.d., packed in-house with 1.9 μm C18 resin from Dr. Maisch GmbH) using an EASY-nanoLC 1200 system equipped with an autosampler.

  • a.
    Use mobile phases consisting of (A) 0.1% FA; (B) 0.1% FA, 80% ACN.
  • b.
    Separate peptides using a 90-min gradient at a constant flow rate of 300 nL/min as follows: 0–4 min, 3%–8% B; 4–67 min, 8%–28% B; 67–79 min, 28%–40% B; 79–84 min, 40%–50% B; 84–87 min, 50%–100% B; and 87–90 min, 100% B.
• 60.
  Analyze the eluting peptides online using a Q-Exactive HFX mass spectrometer via electrospray ionization under the following parameters:

  • a.
    Acquire the data in data-dependent acquisition mode (top-40).
  • b.
    For MS1, set the scan range to 300 - 1650 m/z with a resolution of 60,000. Set the automatic gain control (AGC) target to 3e6, with a maximum injection time of 20 ms.
  • c.
    For MS2, set the resolution to 15,000. Set the AGC target to 1e5, with a maximum injection time of 25 ms.
  • d.
    Set the isolation window to 1.4 m/z. Fragment precursor ions using higher-energy collisional dissociation (HCD) with a normalized collision energy of 27%.
  • e.
    Other general parameters are as follows: set the spray voltage (+) to 3,000; set the S-Lens RF level to 60; set the dynamic exclusion to 15 s; set the default charge state to 2⁺ and exclude unassigned, 1⁺, 7⁺, 8⁺ and >8⁺ charge state; set the minimum AGC to 2.5e2, set the intensity threshold to 1e4; set the isolation window to 1.4 m/z.
• 61.Finally, process all acquired raw files using a uniform data analysis workflow as outlined in the following steps.

Note: For each treatment group, analyze the four biological replicates consecutively by LC-MS/MS. To minimize potential column carryover between groups, perform a 30-min wash run at a constant flow rate of 300 nL/min with the following gradient: 0–3 min, 6%–100% B; 3–6 min, 100% B; 6–9 min, 100%–6% B; 9–12 min, 6% B; 12–15 min, 6%–100% B; 15–18 min, 100% B; 18–21 min, 100%–6% B; 21–24 min, 6% B; 24–27 min, 6%–100% B; 27–30 min, 100% B.

Note: The in-house packed C18 analytical column (150 mm × 100 μm i.d., 1.9 μm resin) may be substituted with a commercially available C18 column of comparable dimensions and particle size, although equivalent performance was not formally evaluated in this study.

Data analysis and processing

Timing: ∼1 day

• 62.
  Perform protein identification and quantification using MaxQuant (version 2.0.3.0)⁸ with the following parameters:

  • a.
    Search the tandem mass spectra against the UniProt mouse protein database (containing 17,073 entries; downloaded in May 2021). The software comes with a comprehensive database of common contaminating proteins built-in and enabled by default.
  • b.
    Set Trypsin/P as the enzyme, and set the maximum number of missed cleavages to 2.
  • c.
    Set cysteine carbamidomethylation (+57.021 Da) as a static modification and set methionine oxidation (+15.995 Da) and protein N-terminal acetylation (+42.010 Da) as variable modifications.
  • d.
    Set the minimum peptide length to 7 amino acids and the maximum peptide mass to 4600 Da.
  • e.
    Set both the precursor and fragment mass tolerances to 20 ppm.
  • f.
    Control the false discovery rates at both the peptide-spectrum match and protein levels at <1%.
  • g.
    Set the minimum number of unique plus razor peptides required for protein identification to 1.
  • h.
    Quantify protein abundance using MaxQuant protein intensity values, which are derived from MS1 precursor ion intensities of all unique and razor peptides assigned to each protein group.
  • i.
    Enable the match between runs (MBR) option using the default matching and alignment time windows.
• 63.
  Perform data processing using Perseus (version 2.0.7.0)⁹ with the following parameters:

  • a.
    First, perform quality control by filtering the identified proteins to retain only those consistently detected in at least three independent replicates.
  • b.
    Then normalize the dataset using the abundance of streptavidin, which is immobilized on the agarose beads used for probe pull-down and can be digested during sample preparation for mass spectrometry.
  • c.
    Subsequently, impute missing values with random values drawn from a downward-shifted distribution to facilitate statistical analysis. Apply a down shift of 1.8 and a distribution width of 0.5 to simulate the abundance levels of low-abundance proteins that typically result in missing values.
  • d.
    Export the imputed data to an Excel spreadsheet for statistical analysis.
  • e.
    Calculate P values (two-tailed Student’s t-test, unadjusted p-value) and fold changes for comparisons between groups (e.g., probe vs. spermidine; probe vs. probe + spermidine; WT+probe vs. DHPS-KO+probe).
  • f.
    Consider proteins with a fold change > 2 and p < 0.05 as significantly differentially labeled protein targets.
  • g.
    Define candidate hypusinated proteins as those specifically labeled by alkyne–spermidine probe in wild-type (WT) cells and dependent on DHPS activity.

Note: Probe-specific labeling in WT cells is defined as proteins significantly enriched in the presence of the probe compared with spermidine control (probe vs. spermidine; fold change > 2; P < 0.05), with signal significantly reduced upon competition with excess native spermidine (probe vs. probe + excess spermidine; fold change > 2; P < 0.05). In parallel, DHPS dependence is defined as proteins significantly enriched in WT cells compared with that in DHPS knockout (DHPS KO) cells under the probe labeling condition (WT+probe vs. DHPS KO+probe; fold change > 2; P < 0.05). Only proteins that meet all of the above criteria were defined as candidate hypusinated proteins.

Note: If NEM is included in the lysis buffer in step 4, set both carbamidomethylation (+57.021 Da) and NEM modification (+125.048 Da) on cysteine as variable modifications during the database search in step 62c.

Note: Protein quantitative abundances were first transformed to log2 scale prior to imputation using the normal distribution parameters described in step 63c.

Expected outcomes

The execution of this protocol is expected to yield a high-purity alkynyl-spermidine probe, confirmed by NMR spectroscopy showing characteristic peaks for the propargyl group and the removal of Boc protecting groups. In western blot analysis, researchers should observe a robust pattern of protein labeling in wild-type (WT) MEFs treated with the probe, visualized via streptavidin-HRP (Figure 6). This labeling should be significantly outcompeted by natural spermidine and markedly reduced in DHPS-knockout (KO) cells, confirming the biological specificity of the metabolic labeling.

Figure 6.

Validation of probe-labeled proteins by western blot analysis

The specificity of the clickable alkynyl-spermidine probe was assessed in wild-type (WT) and DHPS knockout (DHPS-KO) mouse embryonic fibroblasts (MEFs). Cells were pretreated with 20 μM MDL-72527 for 1 h to inhibit polyamine oxidation and improve intracellular probe stability, followed by treatment with 400 μM probe for 4 h, with or without 2 mM competing natural spermidine. Following cell lysis in 1% SDS containing proteinase inhibitor cocktail, and NEM, the lysates underwent CuAAC-mediated click chemistry with azide-biotin. Biotinylated proteins were enriched using streptavidin agarose beads, subjected to SDS-PAGE, and visualized via streptavidin-HRP blotting.

For the proteomic workflow, the on-bead digestion and subsequent LC-MS/MS analysis are expected to identify hundreds of potential hypusination substrates. Successful data processing using MaxQuant and Perseus should reveal significantly enriched proteins, such as eIF5A and RIPK1, with a fold change >2 and p < 0.05 when comparing probe-treated groups to controls. Additionally, endogenous biotinylated proteins such as pyruvate carboxylase and acetyl-CoA carboxylase 1^10,11 should serve as reliable internal recovery markers and show no differential enrichment between conditions.

Limitations

This protocol does not directly identify hypusination sites on target proteins. Instead, it enables the identification of candidate hypusinated proteins based on competitive labeling and comparison with DHPS knockout controls. As such, the approach cannot unambiguously distinguish bona fide hypusinated substrates from proteins indirectly associated with spermidine probe incorporation. Definitive site-specific validation requires additional experiments, including enrichment of individual proteins of interest followed by mass spectrometry–based site identification, in vitro hypusination assays to confirm modification sites, and the generation of site-specific antibodies for orthogonal validation. In addition, the metabolic labeling strategy may introduce biases related to probe uptake, intracellular availability, and competition with endogenous spermidine. Furthermore, transient or low-stoichiometry hypusination events may escape detection. Finally, because this workflow relies on cell-based labeling, its applicability to certain cell types or in vivo systems may be limited and require further optimization.

Troubleshooting

Problem 1

Low yield or purity of the alkynyl-spermidine probe (Related to steps 1–3 in preparation of the clickable alkynyl-spermidine probe).

Potential solution

Ensure all anhydrous solvents are strictly dry and reagents are added slowly in an ice bath to prevent side reactions. Verify intermediate purity by NMR before proceeding to the global deprotection step.

Problem 2

Inconsistent knockout efficiency in MEFs (Related to step 4-7 in preparation of the DHPS knockout MEFs).

Potential solution

Use low-passage MEFs for transduction and confirm DHPS depletion via western blot prior to starting the metabolic labeling experiments.

Problem 3

Weak or absent labeling signal in western blot (Related to steps 1-19 in click chemistry reaction and streptavidin pull-down).

Potential solution

Check the activity of the CuAAC reagents; prepare CuSO₄ and TCEP freshly for every experiment. Ensure the protein concentration is at least 1 mg/mL to facilitate efficient reaction kinetics.

Problem 4

High non-specific background in pull-down samples (Related to steps 1-19 in click chemistry reaction and streptavidin pull-down).

Potential solution

Strictly adhere to the washing protocol, using 1% SDS to remove non-covalently bound proteins from the streptavidin beads.

Problem 5

Poor peptide recovery after on-bead digestion (Related to steps 32–54 in on-bead digestion of biotin pull-down samples, and desalting).

Potential solution

Confirm the urea concentration is diluted to 2 M or lower before adding trypsin, as high urea concentrations inhibit enzymatic activity.

Problem 6

Column clogging or high pressure during LC separation (Related to steps 55-61 in LC-MS/MS data acquisition).

Potential solution

Perform high-speed centrifugation (≥13,000 × g, 4 °C, 15 min) before injection. Carefully transfer only the clear supernatant. Optionally, filter samples using a 0.22 μm filter prior to LC loading.

Problem 7

Poor chromatographic separation or unstable retention time (Related to steps 55-61 in LC-MS/MS data acquisition).

Potential solution

Ensure proper preparation and degassing of mobile phases. Include blank or wash runs between different sample groups to minimize carryover. Replace the analytical column if performance declines.

Problem 8

Low identification rate or high missing values in MaxQuant analysis (Related to steps 62-63 in Data analysis and processing).

Potential solution

Verify mass accuracy and calibration of the instrument. Ensure correct database selection and parameter settings (enzyme specificity, modifications, FDR). Increase LC gradient length or sample loading to improve peptide coverage. Consider increasing the number of biological or technical replicates.

Problem 9

Failure to identify candidate hypusinated proteins (Related to steps 62-63 in Data analysis and processing).

Potential solution

Confirm effective competition conditions (e.g., sufficient spermidine concentration). Validate DHPS knockout efficiency by western blot prior to proteomics. Increase replicate number to improve statistical robustness. Re-evaluate fold change and significance thresholds if overly stringent.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Junying Yuan (junying_yuan@sioc.ac.cn).

Technical contact

Technical questions should be directed to and will be fulfilled by the technical contact, Bing Shan (shanbing@sioc.ac.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or codes.

Acknowledgments

This project was supported (to J.Y.) by the National Natural Science Foundation of China (82188101), Brain Science and Brain-like Intelligence Technology-National Science and Technology Major Project (2025ZD0214800), the Shanghai Basic Research Pioneer Project, the Shanghai Municipal Science and Technology Major Project and the Shanghai Key Laboratory of Aging Studies (19DZ2260400), and by the National Natural Science Foundation of China (32370796 to B.S.).

Author contributions

J.Y., B.S., and T.Z. conceived of this project. T.Z. and J.L. performed the majority of the experiments. Y.Z., Z.-J.Z., and B.S. supervised J.L. and X.Z. in performing the mass spectrometry experiments. Z.W. supervised X.H. in the probe synthesis. L.T. supervised H.Z. in NMR analysis. J.Y., B.S., T.Z., J.L., and X.H. wrote the paper.

Declaration of interests

The authors declare no competing interests.