Genetically encoding ε-N-methacryllysine into proteins in live cells

Reagents and strains

Unless otherwise noted, all commercial reagents were used directly without further purification. The following antibodies were used: anti-6×His-tag (Mouse, Proteintech, cat. no. HRP-66005, 1:10,000), anti-FLAG tag (Mouse, HUABIO, cat. no. M1403-2, 1:10,000), anti-Strep-tag II (Mouse, Wuhan Dian Biotechnology, cat. no. 2098, 1:10,000), Goat anti-Mouse IgG (H+L) HRP conjugate (Jackson ImmunoResearch, Code. 115-035-003, 1:10,000), Goat anti-rabbit IgG (H+L) HRP conjugate (Jackson ImmunoResearch, Code. 115-035-003, 1:10,000), anti-Methacryllysine (Mouse, PTO BIO, cat. no. PTM-1501, 1:2000), anti-Crotonyllysine (Mouse, PTO BIO, cat. no. PTM-502, 1:2000), nti-β-Tubulin (Mouse, Abmart, cat, no. M20005, 1:5000), anti-V5 tag (Mouse, Thermo, cat. no. MA5-15253, 1:2000), anti-Cyclophilin A (Rabbit, HUABIO, at. no. ET1703-33,1:5000). The DH10B strain was laboratory stored. It was used for plasmid amplification and protein expression. HEK293T and HeLa cells were obtained from the American Type Culture Collection (ATCC).

Plasmid construction

To incorporate MeaK in E. coli, the pSupAR-MbPylRS-MbtRNA^pyl_CUA plasmid was constructed by introducing Y349F mutation to MbPylRS. The genes encoding target proteins including EGFP and MBP-Z were cloned into the pBAD vector with a His-tag. The Amber stop codon TAG was introduced at the incorporation site by site-directed mutagenesis.

To incorporate MeaK and CrK in mammalian cells, the pNEU-chPylRS-MmtRNA^pyl_CUA plasmid was constructed by introducing Y384F mutations into the chimeric pyrrolysyl-tRNA synthetase. The genes encoding target proteins including His-tagged EGFP, TXN1 and H3, CypA with Strep-tag II, were cloned into pRK5M vector. All other eukaryotic expressed proteins carried a Flag-tag at the carboxyl terminus were cloned into the pRK5M vector. The Amber stop codon TAG or other mutations were introduced by site-directed mutagenesis.

Recombinant protein expression

For prokaryotic expression, the plasmid pBAD-MBP-Z-E24TAG was co-transformed with pSupAR-MbPylRS Y349F-MbtRNA^pyl_CUA into DH10B competent cells. The transformed cells were plated on LB (Luria-Bertani) agar plate with ampicillin (Sangon) and chloramphenicol (Sangon) and incubated overnight at 37 °C. A single colony was picked and incubated with 1 mL LB medium, which was further diluted into 50 mL LB. When OD600 reached 0.6–0.8, the cell culture was induced with 0.2% arabinose in the absence or presence of 1 mM MeaK. After incubation at 30 °C for 16 h, cell pellets were collected by centrifugation at 4000 × g for 30 min at 4 °C and stored at −80 °C.

For eukaryotic expression, HEK293T cells were seeded in two 10cm-cell culture dishes containing 8 mL of DMEM media with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin–streptomycin (P/S), and grown at 37 °C in a CO₂ incubator. The plasmid pNEU-chPylRS Y384F-MmtRNA^pyl_CUA was co-transfected with pRK5M-EGFP Y151TAG, pRK5M-CypA K125TAG or pRK5M-CypA K131TAG into target cells with polyethyleneimine (1 mg/mL) in DMEM media, separately. Six hours post transfection, the cells were treated with or without 1 mM MeaK and cultured for 36 h. After incubation, the cells were washed with PBS and cell pellets were collected by centrifugation at 500 × g for 5 min at 4 °C and stored at -80 °C.

Protein purification

For the purification of His-tagged MBP-Z E24MeaK, cell pellets were resuspended in His-tag lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 1% v/v Tween 20, and protease inhibitors). The cell suspension was lysed by sonication (Fisher Scientific, φ6, 30% output, 10 min, 5 s off, 3 s on) followed by centrifugation (16,200 × g, 30 min, 4 °C). The supernatant was incubated with 200 μL pre-equilibrated Ni-NTA Beads (Smart-Lifesciences) at 4 °C for 2 h. Then the beads were washed with three volumes of wash buffer 1 (50 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole) and wash buffer 2 (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM imidazole). The proteins were eluted with His-tag elution buffer (50 mM Tris pH 8.0, 500 mM NaCl, 250 mM imidazole), and the buffer was exchanged to storage buffer (50 mM HEPES, pH 7.5, 150 mM NaCl).

For the purification of CypA K125MeaK and CypA K131MeaK with Strep-tag II, cell pellets were resuspended in Strep-tag lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and protease inhibitors). The cell suspension was lysed by sonication (φ3, 30% output, 3 min, 5 s off, 3 s on) followed by centrifugation (16,200 × g, 30 min, 4 °C). The supernatant was incubated with 200 μL pre-equilibrated Streptactin Beads 4FF (Smart-Lifesciences) at 4 °C for 2 h. Then the beads were washed with six volumes of wash buffer I (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA) and eluted with Strep-tag elution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 20 mM D-biotin), followed by buffer exchanging to storage buffer (50 mM HEPES, pH 7.5, 150 mM NaCl).

Genetic incorporation of MeaK

In mammalian cells, HEK293T cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS and 1% P/S. pNEU-chPylRS Y384F-MmtRNA^pyl_CUA and pRK5M-EGFP Y151TAG were co-transfected at 70% cell confluency. Six hours post transfection, the media containing transfection complex was replaced with fresh DMEM media with 10% FBS and 1% P/S in the presence or absence of 1 mM MeaK. After incubation at 37 °C for 36 h, transfected cells were imaged. For optimization of MeaK concentration, cells were cultured under different concentrations of MeaK for 36 h for imaging.

In E. coli, pBAD-EGFP D190TAG-His-tag was co-transformed with pSupAR-MbPylRS Y349F-MbtRNA^pyl_CUA into DH10B. E. coli chemocompetent cells were seeded on LB agar plates containing ampicillin and chloramphenicol. A single colony was picked and incubated into 1 mL 2× YT medium with ampicillin and chloramphenicol at 37 °C. When OD600 reached 0.6–0.8, 500 μL cell culture was supplemented with 1 mM MeaK and 0.2% arabinose, then incubation at 30 °C for 16 h. As a negative control, 500 μL cell culture was induced with 0.2% arabinose at 30 °C for 16 h. Cell pellets were collected by centrifugation at 4000 × g for 10 min. The cells were then rinsed with 2× SDS-PAGE sample loading buffer. The samples were separated on Tricine-SDS-PAGE and immunoblotted with 1:10000 HRP-conjugated 6×His monoclonal antibody.

Flow cytometric analysis of ncAA incorporation in mammalian cells

HEK293T cells were seeded in a 12-well cell culture dish containing 1 mL of DMEM media with 10% FBS and 1% P/S, and grown at 37 °C in a CO₂ incubator. The plasmid pNEU-chPylRS Y384F-MmtRNA^pyl_CUA was co-transfected with pRK5M-EGFP (Y151TAG) into target cells with polyethyleneimine (1 mg/mL) in DMEM media. Six hours post transfection, the cells were treated with or without 1 mM MeaK and cultured for 36 h. After incubation, the cells were washed with PBS and resuspended in 500 µL PBS. Flow cytometry was performed with Beckman CytoFlex and data analysis and plotting were performed with FlowJo (10.8.1).

Fluorescence response of EGFP K85TAG

HEK293T cells were seeded in a 12-well cell culture dish containing 1 mL of DMEM media with 10% FBS and 1% P/S and grown at 37 °C in a CO₂ incubator. The plasmid pNEU-chPylRS Y384F-MmtRNA^pyl_CUA was co-transfected with pRK5M-mCherry-P2A-EGFP (K85TAG) into target cells with 3 µL polyethyleneimine in 1 mL DMEM media. mCherry proteins were used as an internal control, and the self-cleavable peptide allows mCherry and EGFP to separate and fold independently after translation. Six hours post transfection, the media were replaced with complete DMEM media with or without 1 mM MeaK, 10 mM NAM and 10 mM NaBu. After incubation for 36 h, the cells were imaged with a microscope. The total intensity of EGFP and mCherry were measured separately using ImageJ software and the signal of EGFP was normalized to the mCherry signal for each group. The normalized data was further analyzed by 2-tailed student’s t-test and plotted with Graph Prism 9.0.

Co-immunoprecipitation (Co-IP)

HEK293T cells were harvested after transfection for 48 h, the cells were washed 2 times with PBS, resuspended with 1 mL PBS and centrifuged (500 × g, 4 °C, 3 min) to remove the upper liquid. The samples were resuspended with 500 µL Strep-tag lysis buffer, and sonicated with Sonic Dismembrator (Fisher Scientific, φ3, 30% output, 1 min, 5 s off, 3 s on) in an ice-water bath, followed by centrifugation (21,000 × g, 30 min, 4 °C). The soluble fractions were collected and incubated with pre-equilibrated Streptactin Beads 4FF at 4 °C for 1 h with constant mechanical rotation. Then centrifuging and removing the upper liquid, and washing the samples 3 times by wash buffer I and 3 times by wash buffer II (100 mM Tris-HCl pH 8.0, 37.5 mM NaCl, 1 mM EDTA). 4× protein loading was added for Western blot analysis. The samples were separated on 10% Tricine-SDS-PAGE and immunoblotted with specific primary antibody solution overnight at 4 °C. After incubation with the matching secondary antibody, chemiluminescent detection was performed using enhanced chemiluminescence (ECL) luminous solution (Vazyme).

Mass analysis of intact proteins

Purified proteins were analyzed on mass spectra of intact proteins obtained using a Triple TOF 6600 (SCIEX) MS System equipped with an electrospray ionization (ESI) source in conjunction with SCIEX Analyst TF software. Protein samples were separated and desalted on a Aeris 3.6 µm WIDEPORE C4 LC Column (200 Å, 2.1 × 50 mm, 3.6 μm, Phenomenex). Solvent A was 0.1% formic acid in water and solvent B was acetonitrile. A constant flow rate of 0.3 mL/min was used. Mass spectral deconvolution was performed using SCIEX OS-Q software (version 2.0, SCIEX Corporation).

Sample preparation for in-solution digestion

Purified proteins were precipitated with 6 times the volume of 100% acetone 2 h at −20 °C. The protein pellets were washed twice with ice-cold acetone and air-dried. The protein pellets were resuspended in 50 mM ammonium bicarbonate and digested with trypsin (1:50) overnight at 37 °C. Then, peptides were reduced with 5 mM tris-carboxyethylphosphine (TCEP) and alkylated with 10 mM iodoacetamide (IAA) in the dark for 1 h at room temperature. Then, stopped by acidification with formic acid (FA) at a final concentration of 5% (v:v). Peptides were desalted with StageTip and dried.

Affinity purification and mass spectrometry (AP-MS)

As described previously in Co-IP assay, after washing with buffer I and II, beads were resuspended in 40 μL 8 M urea in Tris buffer. Protein was reduced with 0.4 μL of 500 mM TCEP for 15 min at room temperature before adding 0.8 μL of 550 mM IAA for 20 min at room temperature in the dark and followed by dilution to 2 M urea with 100 mM pH 8.5 Tris buffer. Proteins were digested on the beads with 1.5 μL 1 μg/μL Trypsin (YaxinBIO) at 37 °C in the dark overnight. The digestion stopped by adding FA (Formic acid) to a final concentration of 5%. The supernatant without beads was transferred to a new tube and was collected in three repetitions with 5% FA. Peptides were desalted using StageTip and dried.

HPLC-MS/MS analysis

Peptides were redissolved in 0.1% FA and separated on C18 (75 μm × 15 cm, 1.9 µm C18, 1 µm tip) with Easy-nLC 1200 system (Thermo Fisher Scientific). Using the following mobile phases: 2% ACN (Acetonitrile) incorporating 0.1% FA (Solvent A) and 80% ACN incorporating 0.1% FA (Solvent B). Samples were analyzed with a 60 min gradient at a flow rate of 450 nL/min. Peptides were ionized by electrospray ionization at +2.1 kV. Tandem mass spectrometry analysis was carried out on a Q‐Exactive HF-X mass spectrometer (Thermo Fisher Scientific) under the control of Xcalibur software (version 4.5.445.18). The mass spectrometer operated in data-dependent acquisition mode, and survey scans were obtained in a full mass scan range of 400–1400 m/z with lock mass activated. In each scan cycle, fragmentation spectra of the 20 most intense peptide precursors in the survey scan were acquired in the higher-energy collisional dissociation (HCD) mode. Full MS scan at resolution of 60,000, followed by MS/MS scans at resolution of 15,000 with an isolation width of 1.6 m/z. The AGC targets for MS1 and MS2 scans were 3 × 10⁶ and 1 × 10⁵, respectively, and the maximum injection time for MS1 and MS2 were 45 and 22 ms, respectively. Precursors of +1, +6, +8 or above, or unassigned charge states were rejected; exclusion of isotopes was disabled; dynamic exclusion was set to 30 s. Raw files of AP-MS were collected as three biological replicates. Other LC-MS/MS experiments were performed once.

MS data analysis

Modified peptides were identified using pFind software. pFind (version 3.2.0) search parameters: precursor mass tolerance 20 parts per million (ppm); fragment mass tolerance 20 ppm; peptide length minimum 6 amino acids and maximum 100 amino acids per chain; peptide mass minimum 600 and maximum 10,000 Da per chain. Carbamidomethyl[C] (+57.0215 Da) was the fixed modification; Acetyl[AnyN-term] (+42.0106 Da) and oxidation[M] (+15.9949 Da) were the variable modification; Trypsin as the digestive enzyme; three missed cleavage sites per chain. maximum false discovery rate (FDR) for peptides and proteins of 1%. The human protein sequences were downloaded from Uniprot. The “Open Search” mode was activated when unknown modifications were present on the peptide.

For AP-MS raw data was processed in the MaxQuant software (version 2.0.3.0), The minimal required peptide length was set to seven amino acids and both protein and peptide identifications were accepted at a FDR of 1%. Database searches were performed according to standard settings with trypsin being used, allowing 2 missed cleavages. Oxidation [M], Acetyl [Protein N-term] and Kmea[K] (+68.023 Da) were allowed as variable modifications with a maximum number of 5. Carbamidomethyl [C] was allowed as a fixed modification. The match between runs feature and the label-free quantitation function were activated. All other parameters were left at default.

Microscale thermophoresis (MST) binding assay

Purification of recombinant EGFP fusions of CypA and CypA K125MeaK. Analysis was performed in buffer (50 mM HEPES, 150 mM NaCl and 5% DMSO). 5 μL of target protein was mixed with 5 μL of 2-fold serially diluted CsA at room temperature. Mixture samples were obtained through Monolith Capillaries (Catalog No.MO-K022). Signal was measured by Monolith NT.115 (NanoTemper) with Nano BLUE detector. Curve fitting was performed by using MO.Affinity Analysis v3.0.6. Data are presented as mean ± SD. The given Kd values were calculated with a 95% confidence level from three experiments.

Flow cytometric analysis of ROS level in HeLa cells

HeLa cells were seeded in a 12-well cell culture dish containing 1 mL of DMEM media with 10% FBS and 1% P/S, and grown at 37 °C in a CO₂ incubator overnight. The plasmid pRK5M-mCherry, pRK5M-CypA-WT-mCherry, pRK5M-CypA-K125TAG-mCherry and pNEU-chPylRS Y384F-MmtRNA^pyl_CUA were transfected into target cells with 3 µL PEI in 1 mL DMEM media. 6 h post transfection, the media were replaced with complete DMEM media with or without 1 mM MeaK and 4 µM CsA. After incubation for 36 h, the cells were washed 2 times with PBS. Then the cells were incubated with intracellular ROS probe DCFH-DA (OwowLife, 10 µM) for 20 min at 37 °C, and washed 2 times with PBS. The cells were then rinsed with 500 µL PBS and analyzed by Beckman CytoFlex. Positive cells (n = 5000) with the mCherry fluorescence signal were collected and then the mean FTIC-A intensity of positive cells was measured by CytoExpert software, which indicates the level of intracellular ROS, and was plotted with FlowJo 10.8.1.

In vitro and in vivo demethacrylation assay

In vitro, 400 ng purificated recombinant HDAC1 protein and 1 µg CypA K125MeaK protein was incubated in reaction buffer for 2 h at 30 °C. The reaction buffer for the deacylation was 25 mM Tris-HCL, with 150 mM NaCl, 2 mM MgCl₂, 1 µM ZnCl₂, 1 mM DTT. Protein loading was added for the next Western blot analysis.

In vivo, HEK293T cells were seeded in a 12-well cell culture dish. The plasmids pNEU-chPylRS Y384F-MmtRNA^pyl_CUA and pRK5M-CypA-K125TAG-Strep were co-transfected with or without plasmid pRK5M-HDAC1-Flag into target cells with 3 µL polyethyleneimine in 1 mL DMEM media. Six hours post transfection, the media were replaced with complete DMEM media with 1 mM MeaK.Then HEK293T cells were harvested after transfection for 48 h. The samples were resuspended with 500 µL Strep-tag lysis buffer, and sonicated in an ice-water bath, followed by centrifugation (21,000 × g, 30 min, 4 °C). Soluble fractions were collected and incubated with pre-equilibrated Streptactin Beads 4FF at 4 °C for 1 h with constant mechanical rotation. Then centrifuging and removing the upper liquid, and washing the samples 3 times by wash buffer I and 3 times by wash buffer II. Protein loading was added for the next Western blot analysis.

Bioinformatics analysis

Unless noted, normalization of raw read counts was done using the preprocessCore package (v.4.6.0) in R. Raw data results of maxquant output were normalized by the quantile normalization function embedded in preprocessCore, finally, the protein expression values were presented in the log2. Principal component analysis (PCA) of the data matrix using the prcomp function in R. Proteins were generated using the normalized raw data excluding individuals with protein expression deficiencies. The proteins were ranked from largest to smallest among individuals using the coefficient of variation, and the top 1000 proteins were taken as input to apply principal component analysis.

GO analysis of genes with differentially expressed proteins was performed by the R package ClusterProfiler package (v.4.0.5), a tool that analyzes functional profiles of gene and gene clusters. The protein IDs included in UniProt were converted to the corresponding gene Entrez IDs. GO terms with p-value 

Statistics and reproducibility

The experiments in the Figs. 2c; 5e–g were repeated twice with similar results.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.