Bioorthogonal probe

In Raw264.7 cell lysates, itaconate-alkyne (ITalk; 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mM; 0–4 hours) exhibits concentration- and time-dependent labeling [1]. For four hours, itaconate-alkyne (100 μM) dramatically boosted the protein level of NRF-2 in Raw264.7 cells stimulated with 100 ng/mL of LPS and significantly reduced the number of lactate-producing cells in LPS-stimulated Raw264.7 cells. LPS-stimulated Raw264.7 cells secrete considerably less interleukin 1β (IL-1β) when itaconate-alkyne is present [1]. Itaconate-alkyne (100 μM; 12 hours) has no effect on Raw264.7 cell viability [1]. In inflammatory macrophages, itaconate-alkyne binds to its targets, which include adenylate kinase 2 (AK2), gelsolin (GSN), ATP citrate synthase (ACLY), and DNA damage binding protein 1 (DDB1)[1].

In vitro proteins labeling by itaconate-alkyne (ITalk) . [1]
For the labeling of proteins in cell lysates, frozen Raw264.7 cells were resuspended in ice-cold PBS buffer containing EDTA-free Pierce HaltTM protease inhibitor cocktail. The cells were lysed by sonication in ice and cell lysates were collected by centrifugation (20,000 g, 30 min) at 4 C to remove the debris. The protein concentration was determined by using the BCA protein assay kit. 50 µL of cell lysates (2 mg/ml) were incubated with 100 μM itaconate-alkyne (ITalk) at 37 C for 1 h. The resulting lysates were precipitated by 200 µL methanol, 50 µL chloroform and 150 µL Milli-Q water. The precipitated proteins were centrifuged at 8000 g for 5 min at 4 C and washed twice with 500 µL cold methanol. For visualizing the probe labeling efficiency by in-gel fluorescence, the precipitated proteins were resuspended in 50 µL PBS containing 0.4% SDS, 1 mM CuSO4, 100 μM TBTA ligand, 100 μM Rhodamine-azide, and 1 mM TCEP for 1 h at room temperature. The reacted samples were resolved on 10% SDS-PAGE gels and imaged by ChemiDoc XRS+. The gels were then stained by Coomassie brilliant blue to demonstrate equal loading.

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In situ proteins labeling by itaconate-alkyne (ITalk) . [1]
For the labeling of itaconate targets in living cells, the Raw264.7 cells were grown to 80% confluence. The cells were treated with 100 μM OI or itaconate-alkyne (ITalk) for 12 h. The cells were washed with PBS for three times and centrifuged at 1000 rpm for 3 min. The cell pellets were stored at -80 °C. The cell pellets were resuspended in ice-cold PBS buffer containing EDTA-free Pierce HaltTM protease inhibitor cocktail. The cells were lysed by sonication in the ice and cell lysates were collected by centrifugation (20,000 g, 30 min) at 4 C to remove the debris. The protein concentration was determined by using the BCA protein assay kit. For visualizing the probe labeling efficiency by in-gel fluorescence, 50 μL of lysates (2 mg/mL) were mixed with 1 mM CuSO4, 100 μM TBTA ligand, 100 μM Rhodamine-Azide, and 1 mM TCEP for 1 h at room temperature. The reacted samples were resolved on 10% SDSPAGE gels and imaged by ChemiDoc XRS+. The gels were then stained by coomassie brilliant blue to demonstrate equal loading.
Time-resolved chemoproteomics for identification of sensitive itaconation targets. For the time-resolved chemoproteomic profiling, Raw264.7 cells were grown to 80% confluence in 15 cm dish. The cells were treated with 10 ng/mL LPS and 100 μM itaconate-alkyne (ITalk) for 1 or 10 h, respectively. The procedures for cell lysis, click reaction, streptavidin enrichment and trypsin digestion were performed as described above. For dimethyl labeling, the samples from 1 h treatment were labeled as “light” while those from 10 h treatment were labeled as “heavy”. The data analysis was performed as described above.

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Identification of itaconation sites by itaconate-alkyne (ITalk) . [1]
For the identification of itaconation sites by itaconate-alkyne (ITalk) , Raw264.7 cells were grown to 80% confluence in 15 cm dish. Raw264.7 cells were treated with 10 ng/mL LPS and 100 μM itaconate-alkyne (ITalk) for 12 h. The cells were collected by PBS wash for three times and centrifugation at 1000 rpm for 3 min. The cells were lysed in 1 mL ice-cold PBS buffer containing EDTA-free Pierce HaltTM protease inhibitor cocktail with sonication. The cell lysates were collected by centrifugation (20,000 g, 30 min) at 4 C to remove the debris. The protein concentration was determined by using the BCA protein assay kit. 1 mL cell lysates (2 mg/mL) were reacted with 1 mM CuSO4, 100 μM TBTA ligand, 100 µM acid-cleavable azidebiotin (DADPS Biotin Azide, cat. no. 1330-5), and 1 mM TCEP for 1 h at room temperature. The resulting click-labeled lysates were centrifuged at 8000 g for 5 min at 4 C and washed twice with 1 mL cold methanol. The streptavidin enrichment and on-beads Trypsin digestion were performed as described above. Release of the modified peptides from the beads was carried out by incubating the beads with 200 μL of 2% formic acid/water for 1 h with gentle rotation. After centrifugation, the supernatant was collected. Then the cleavage process was repeated and supernatant was combined. In addition, the beads were washed with 50% acetonitrile/water containing 1% formic acid (400 μL), and the washes were combined with the supernatant to form the cleavage fraction. Sample was dried in a vacuum centrifuge and stored at -30 °C until analysis.

Cell viability assays. [1]
For evaluation of the cell toxicity of itaconate-alkyne (ITalk) , 10,000 Raw264.7 cells per well were seeded in 96-well dishes to grow overnight. Then the cells were treated with 100 μM itaconate-alkyne (ITalk) or OI for 24 h. The cells were washed with pre-warmed PBS and incubated with serumfree medium containing MTS reagent for 2h. The absorbance at 490 nm was measured and cell viability under test conditions was reported as a percentage relative to the negative control treatment. For evaluation of the impact of itaconate on necroptosis, 10,000 HT-29 cells per well were seeded in 96-well dishes to grow overnight. The cells were treated with 250 μM OI for 24 h and then treated with 20 ng/mL TNF-α, 1 μg/mL SMAC mimetic and 50 μg/mL zVAD-FMK for 3 h. The cell viability was determined by MTS as described above. For evaluating the cell viability by CellToxTM Green Cytotoxicity Assay, the cells were treated with 100 μL of Green Reagent (2X) per well and incubated at r.t. for 1 h. The fluorescence at 485nmEx/520nmEm was measured for cell viability calculation.

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Quantification of intracellular itaconate. [1]
HEK293T cells were plated at 2 x 106 per well in 6-well plates overnight, and treated with 1mM itaconate-alkyne (ITalk) or OI for 4 hours, respectively. The cells were washed with PBS gently in the plates for three times and collected by centrifugation. The cells were further washed with PBS for three times by centrifugation. The cell pallets were lysed by sonication in ice-cold PBS containing 0.1% TritonX-100, centrifuged at 20000 g for 30 min to remove cell debris, and protein concentrations were determined by BCA protein assay. After normalizing the protein concentration to 2 mg/ml of 100 µL, 900 µL cold methanol was added to extract the small molecule metabolites on ice. The mixture was incubated at -20 ℃ for 2 h and was centrifuged at 20000 g for 1 h at 4 ℃. The supernatant was collected and analyzed by LC-SRM. The LC-SRM system is composed of an AB SCIEX 5500 triple-quadrupole mass spectrometer and a SHIMADZU DGU-20A liquid chromatography instrument with an Agilent column. The buffer gradient is 100%-0 Buffer A (100% water, 0.1% formic acid) and 0%-100% Buffer B (100% methanol, 0.1% formic acid) for 10 min. The absolute concentration of itaconate was calculated according to the standard curve of itaconate.

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Measurement of IL‐1 production by enzyme‐linked immunosorbent assay (ELISA). [1]
Raw264.7 cells were initially plated overnight in 6 cm dish. The cells were treated with 10 ng/mL LPS and 1 mM itaconate or 100 μM itaconate-alkyne (ITalk) or 100 μM OI for 24 h. Afterwards, the condition media were collected and cytokine production was assayed using ELISA kits for IL‐1.

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Glucose consumption and lactate production. [1]
Raw264.7 cells were cultured in 96 wells overnight. The cells were treated with 10 ng/mL LPS and 1 mM itaconate or 100 μM itaconate-alkyne (ITalk) or 100 μM OI for 24 h. Glucose consumption in the culture medium were measured using the Glucose (GO) Assay Kit whereas lactate levels in the culture medium were determined using a Lactate Assay Kit.

[1]. Chemoproteomic Profiling of Itaconation by Bioorthogonal Probes in Inflammatory Macrophages. J Am Chem Soc . 2020 Jun 24;142(25):10894-10898.

Itaconate is an anti-inflammatory metabolite involved in pathogen-macrophage interactions, but its mechanism of action has not been fully elucidated. Competitive cysteine profiling has been used to study the reactivity of Itaconate in cell lysates, but there is still a lack of methods for directly analyzing Itaconate targets in living macrophages. In this study, we developed a specific bioorthogonal probe, Itaconate-alkyne (ITalk), for quantitative and site-specific chemical proteomics analysis of Itaconate in inflamed macrophages. ITalk can reproduce the anti-inflammatory properties of Itaconate and can be used for biochemical assessment and proteomics analysis of its direct targets. Our study reveals the wide distribution of Itaconate substrates, providing a versatile tool and comprehensive resource for studying its function. [1] In summary, we developed a specific and cell-permeable bioorthogonal probe, ITalk, for direct chemical proteomics analysis of Itaconate targets in living cells. The probe exhibits anti-inflammatory activity comparable to that of Itaconate and can label the true targets of Itaconate. Using quantitative chemical proteomics, we identified 1,926 Itaconate protein targets in inflammatory macrophages, including 199 highly sensitive targets. In addition, site-specific analysis revealed 1,131 Itaconate-modified cysteine sites, including sites on key proteins involved in inflammatory responses and host defense. The comprehensive database we constructed provides a valuable resource for further research on the biological functions of Itaconate. For example, we found that Itaconate activates the RIPK3 signaling pathway by modifying the C360 site of RIPK3, and this novel link between metabolism and necrosis-apoptosis in immune signaling needs further investigation. From a chemical perspective, given that ITalk may still be hydrolyzed by esterases and its long tail may not fully mimic the role of Itaconate in cells, there is an urgent need to develop more stable Itaconate-modified probes that are closer to the native state. Nevertheless, since ITalk can be easily applied to analyze Itaconate targets in cell types other than macrophages, we envision that this unique chemical tool will reveal more functional relevance of Itaconate. [1]

C13H18O4

238.28

238.12

2454181-83-8

154573778

White to off-white solid powder

1.1±0.1 g/cm3

385.4±32.0 °C at 760 mmHg

141.5±18.6 °C

0.0±1.9 mmHg at 25°C

1.485

3.21

1

4

10

17

322

0

C=C(CC(=O)OCCCCCCC#C)C(=O)O

NPWMZOZWVAFQMP-UHFFFAOYSA-N

InChI=1S/C13H18O4/c1-3-4-5-6-7-8-9-17-12(14)10-11(2)13(15)16/h1H,2,4-10H2,(H,15,16)

2-methylidene-4-oct-7-ynoxy-4-oxobutanoic acid

Itaconate-alkyne; 2454181-83-8; ITalk; 2-methylidene-4-oct-7-ynoxy-4-oxobutanoic acid; 2-methylene-butanedioic acid, 4-(7-octyn-1-yl) ester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 250 mg/mL (1049.19 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.73 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)