Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure

Chemicals

Dulbecco’s modified eagle medium (DMEM), fetal calf serum (FCS), penicillin/streptomycin solution (P/S), 0.05% trypsin–EDTA and phosphate-buffered saline (PBS) were purchased from Gibco by Life Technologies (Karlsruhe, Germany). PromoFectin transfection reagent was obtained from PromoCell GmbH (Heidelberg, Germany). The DNA construct pcDNA3.1V5-HisB_A123 and the HisPur™ Ni–NTA Spin Purification kit were purchased from ThermoFisher Scientific (Darmstadt. Germany). NuPAGE MES SDS running buffer (20x), NuPAGE transfer buffer (20x), 4–12% Bis–Tris gels, polyvinylidene difluoride (PVDF) membranes (0.2 µm pore size) and Dynabeads protein G were purchased from Invitrogen by Life Technologies (Karlsruhe, Germany). Digitionin, tris(2-carboxyethyl)phosphine-hydrochloride (TCEP-HCl), NaCl, dimethyl-pimelimidate-dihydrochloride (DMP), Tween-20, IAA, NH4HCO3, acetonitrile, triethanolamine (TEA), NaN3, trypsin and the corresponding trypsin reaction buffer from the Trypsin Profile IGD kit were obtained from Sigma-Aldrich (Steinheim, Germany). Tris, acetic acid, formic acid (FA ≥ 98%) and NaOCl solution for decontamination (12% Cl2) were obtained from Carl Roth (Karlsruhe, Germany). Threefold deuterated atropine (d3-atr) was from CDN Isotopes (Pointe Claire, Quebec, Canada). Chameleon Duo marker, 4 × protein loading dye, intercept blocking buffer PBS and IR dye 800CW goat anti-mouse antibody were obtained from Licor (Bad Homburg, Germany). PhastGel Blue tablets were from GE Healthcare (Munich, Germany). Methanol was purchased from Merck (Darmstadt, Germany). 1,4-dithiothreitol (DTT) was purchased from Roche (Penzberg, Germany). The primary anit-hTRPA1 antibody ANKTM-1 (C-5) was obtained from Santa-Cruz Biotechnology (Heidelberg, Germany) and anti-6xHis antibody from abcam (Cambridge, UK). SM (purity and integrity were assessed in-house by nuclear magnetic resonance, NMR, spectroscopy) was made available by the German Ministry of Defense.

Cell culture

Human embryonic kidney wildtype (HEK-wt) cells, kindly donated by the Walther-Straub-Institute (Ludwig-Maximilians-University, Munich), were cultured in DMEM containing 10% (v/v) FCS and 1% (v/v) P/S in a humidified atmosphere at 37 °C, 5% (v/v) CO2. For transfection, 4–5 × 106 HEK-wt cells were seeded in a T175 flask. The next day, cells reached approx. 50% confluency and were transfected with pcDNA3.1V5-HisB_A123 using PromoFectin as follows: 10 µg of the DNA construct and 20 µL PromoFectin solution were each mixed with 1 mL DMEM without supplements. Afterwards, 1 mL PromoFectin solution was added to 1 mL DNA solution. The DNA-PromoFectin mix was incubated for 20 min at room temperature (RT). After removal of the cell culture medium, 2 mL of PromoFectin-DNA solution was added to the flask and filled up with 15 mL DMEM and incubated for 72 h. Transfected and thus hTRPA1 overexpressing HEK293 cells are further referred to as HEK-A1 cells.

Cell lysis

A digitonin lysis buffer was used according to Suo et al. (2019). It was freshly prepared and contained 20 mM Tris, adjusted to pH 8.0, 150 mM NaCl, 5 mM TCEP-HCl and 1% (w/v) digitonin. Protease inhibitors and DNase were not part of the lysis buffer. For cell lysis, 2 mL lysis buffer was added to each T175 flask. Cells were incubated at 4 °C for 1 h before being gently detached by scraping. The whole content was transferred to another Eppendorf tube. The mixture was further incubated on ice for 1 h and intermittently vortexed. The mixture was centrifuged at 14,000 RCF for 15 min and supernatants were stored at − 80 °C.

Immobilized metal affinity chromatography

Cell lysates were purified using immobilized metal affinity chromatography (IMAC). The HisPur™ Ni–NTA spin purification kit, containing 1 mL columns, was used according to the manufacturer’s protocol. In brief, the equilibration buffer contained 10 mM imidazole, the wash buffer 25 mM imidazole and the elution buffer 300 mM imidazole. All centrifugation steps were carried out at 700 RCF for 2 min at 4 °C. The column was equilibrated with 2 mL equilibration buffer at 4 °C for 1 h. The column was then centrifuged and the equilibration fraction was collected (F1). The column was washed four-times with 2 mL wash buffer each, centrifuged, and the wash fractions were collected in separate tubes (W1-W4). The His-tagged protein was eluted with two-times 1 mL elution buffer followed by centrifugation and collection of the eluates in separate vials (E1-E2). All samples were stored at − 80 °C.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The loading buffer contained 60 µL of 4 × loading dye mixed with 40 µL DTT (500 mM). HEK-A1 whole cell lysate (15 µL) was mixed with 8 µL of the loading buffer and loaded onto a 4–12% Bis–Tris gel. As a marker, 3 µL Chameleon Duo marker was used. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was run in ice-cold 1 × NuPAGE MES SDS running buffer at constant voltage (200 V) for 40 min. The gel was either stained with Coomassie brillant blue (CBB) or Western blotting was performed.

Staining of proteins

Proteins were visualized in gel using CBB. A CBB stock solution was composed of one PhastGel Blue tablet dissolved in 200 mL H2O/methanol (40:60 v/v). For protein staining, CBB working solution was freshly prepared and contained 6 mL methanol, 12 mL water, 2 mL acetic acid (100%) and 2.2 mL of CBB stock solution (0.2% w/v). After SDS-PAGE, the gel was carefully removed from the chamber and placed in the CBB solution. The gel was incubated for 45 min on a swirl plate until bands were noticeably stained. CBB solution was then removed and the gel was washed twice with water for 15 min followed by additional de-staining overnight in water. The next day, the protein double band considered as hTRPA1 channel was cut out and subjected to nano-LC MS/MS analysis for protein identification.

Western blot analysis

After SDS-PAGE, proteins were transferred from the gel onto a 0.2 µm PVDF membrane using the wet blotting technique. The membrane was activated in methanol for 30 s and blotting was performed using 1 × transfer buffer containing 20% (v/v) methanol at 25 V for 1 h. The membrane was then blocked in PBS blocking buffer for 1 h on an orbital shaker. As primary antibody solution, either 4 µg anti-hTRPA1 (ANKTM1 C-5) or 4 µg anti-His-tag (anti-6xHis) antibody was diluted in 4 mL 0.2% (v/v) Tween-20 in PBS blocking buffer and incubated overnight. The membrane was washed twice for 10 min with a washing buffer containing 0.1% (v/v) Tween-20 in PBS. The commercial solution of the secondary antibody (800CW goat anti-mouse antibody) was diluted 1:7500 in 11 mL 0.2% (v/v) Tween-20 in PBS blocking buffer and the membrane was incubated for 1 h. Afterwards, the membrane was washed twice as described above. Images were recorded with an Odyssey® DLx imaging system using the software Image studio 5.2 (Licor, Bad Homburg, Germany).

Measurement of the intracellular calcium concentration with Fura-2 AM

Fura-2 AM was used according to the manufacturer’s instructions. A stock solution (1 mM) was prepared. Cells were harvested in the same manner as described before (see section: cell culture) and counted using a CASY Cell Counter and Analyzer TT. Approximately 2*106 cells/mL was used and the cell suspension was centrifuged for 5 min at 500 RCF. Afterwards, cells were loaded with Fura-2 AM (cfinal 2 µM) and incubated for 1 h at 37 °C. The cells were then centrifuged (500 RCF, 3 min), rinsed with DMEM, and centrifuged again (500 RCF, 3 min). Cells were resuspended in DMEM before plating 190 µL of cell suspension into each well of a black 96-well plate. In the case of AP18 pre-treatment, cells were resuspended in DMEM with AP18 (final concentrations: 5 μM, 10 μM and 25 μM). The photometer was adjusted to 37.0 °C. The injector was then primed with the before prepared AITC solution (1 mL). DMEM was used as negative control. The excitation wavelengths for Ca2+-bound and Ca2+-free fura-2 AM were set to 340 nm and 380 nm, respectively. The wavelength of maximum emission in both forms was 510 nm. The ratios 510 nm/340 nm and 510 nm/380 nm are proportional to the quantity of Ca2+ present intracellularly. After recording the baseline for 20 cycles (approx. 40 s), 10 μL of the agonist was injected and changes in fluorescence were measured for 150 s. A 340 nm to 380 nm ratio was determined to assess changes in intracellular calcium levels ([Ca2+]i).

Immunomagnetic separation

Preparing IMS of hTRPA1, commercially available Dynabead protein G slurry (1 mL) was transferred into a 5 mL reaction vial. Beads were fixed using a magnet and the supernatant was discarded. Beads were washed three-times with 2 mL PBST (0.05% v/v Tween-20 in PBS, pH 7.4). Afterwards, 4 mL PBST was added to the beads and either 200 µL of anti-His-tag antibody (1 mg/mL) or 1 mL of anti-hTRPA1 antibody solution (200 µg/mL) was added for immobilization. The mixture was incubated on a rolling shaker for 15 min at RT. The supernatant was removed, and beads were washed twice with 200 mM TEA (0.025% w/v NaN3, pH 7.8). Afterwards, beads were incubated with a freshly prepared DMP solution (2 mL, 5.4 mg/mL in TEA solution) on a rolling shaker for 30 min at RT. The supernatant was again removed and 2 mL Tris-buffered saline (TBS) (0.9% w/v NaCl in 20 mM Tris, pH 7.6) was added. The mixture was incubated for 15 min at RT on a rolling shaker. The beads were washed twice with 1 mL PBST. Finally, 950 µL PBST was added to the labeled beads which were stored at 4 °C.

The labeled beads slurry (50 µL) was transferred into a 1.5 mL reaction vial. Beads were fixed with a magnet and the supernatant was discarded. Afterwards, 200 µL cell lysate was added and the mixture was incubated for 2 h at 20 °C. The supernatant was removed, and beads with the bound protein were washed twice with 500 µL PBST, each. Next, 200 µL PBS and 8 µL DTT (20 mg/mL in water) were added to the vial. Samples were incubated for 30 min at 47 °C. Afterwards, 16 µL IAA (40 mg/mL in water) was added following a 30 min incubation at RT in the dark. The protein labeled beads were washed three-times with 250 µL NH4HCO3 (4 mg/mL) and the supernatant was removed. For proteolysis, 25 µL trypsin solution (20 µg/mL in trypsin solubilization reagent) and 50 µL trypsin reaction buffer (40 mM NH4HCO3 in 9% v/v acetonitrile) was added and incubated overnight at 37 °C under gentle shaking. Afterwards, the supernatant was transferred into a 10 kDa ultrafiltration (UF) device for UF (10,000 RCF, 10 min, 15 °C). The retentate was washed twice by UF with 50 µL d3-atr solution (3 ng/mL in 0.5% v/v FA). The filtrates were centrifuged and subjected to μLC-ESI MS/HR MS analysis or stored at − 20 °C.

Exposure of extracted hTRPA1 protein to SM

HEK-A1 cell lysate (200 µL) was incubated with antibody-labeled beads for 2 h for extraction of hTRPA1 by IMS. Beads with bound hTRPA1 were mixed with 195 µL PBS and incubated (1 h, RT) with 5 µL ethanolic SM solution (diluted in ethanol yielding final concentrations of 1 to 10 mM SM). Blanks had a final concentration of 2.5% (v/v) ethanol only. The liquid layer was discarded and IMS was performed as described above (see section: Immunomagnetic separation).

Exposure of cell culture to SM

HEK-A1 cells were exposed to SM as follows: cell culture medium was removed from the T175 flask 72 h after transfection and 6 mL of an ethanolic SM solution diluted in DMEM (final concentrations: 50 µM, 100 µM, 200 µM, 250 µM, 500 µM and 1000 µM) was added to each flask. As a negative control (blank), 2.5% (v/v) ethanol was used. The cells were incubated for 1 h at 37 °C. Due to the detachment of the cells after exposure to SM concentrations above 500 µM, cell-containing supernatants were collected and centrifuged for 5 min at 500 RCF. The cell pellet was washed with PBS. Cells were again centrifuged and 2 mL lysis buffer (see section: Cell lysis) was added. The pellet was resuspended in lysis buffer and incubated for 1 h on ice. The lysate was further incubated for 1 h and vortexed several times. Cell lysates were centrifuged for 15 min at 14,000 RCF and the supernatants were subjected to clean tubes. Lysates were stored at − 80 °C.

µLC-ESI MS/HR MS analysis

A MicroPro pump (Eldex Laboratories, Napa, CA, USA) with an Integrity autosampler and a Mistral column oven (both Spark Holland, Emmen, The Netherlands) were used for chromatography that was online coupled to a QExactive plus Orbitrap mass spectrometer by an HESI II ion source (Thermo Scientific, Bremen, Germany). Eldex MicroPro 1.0.54 software (Eldex Laboratories) was used to control the system (Blum et al. 2020; John et al. 2022a). The Excalibur 4.1 software (Thermo Scientific) was used to manage the MS system. Calibration was done daily using the Pierce LTQ Velos ESI positive ion calibration solution (Thermo Fisher Scientific). The lock masses of protonated ubiquitous molecules (C24H39O4, m/z 391.28429, and C10H16O2NS, m/z 214.08963) were used for internal mass calibration (John et al. 2022a). To identify peptides obtained from proteolysis of the adducted hTRPA1 channel initial MS/HR MS detection was carried out in the ddMS2 approach. Proteome Discoverer Software 2.5.0.400 (Thermo Scientific) was used to analyze the obtained data.

Using the ddMS2 approach, peptides were separated at 45 °C using a binary mobile phase (30 µL/min) of solvent A (0.05% v/v FA) and solvent B (ACN/H2O 80:20 v/v, 0.05% v/v FA) on an Acquity UPLC HSS T3 column (150 × 1.0 mm I.D., 1.8 μM, Waters, Eschborn, Germany) protected by a precolumn (Security Guard Ultra cartridge C18 peptide; Phenomenex, Aschaffenburg, Germany). Solvent A and solvent B were applied in gradient mode: t[min]/B[%] 0/4; 3/4; 60/40; 60.5/95; 68.5/95; 69/4; 70/4 with an initial 15 min equilibration period under starting conditions. Eluates between retention time (tR) 4.5 min and 60 min were directed toward the mass spectrometer using a six-port valve. For full scan MS analysis, resolution was 70,000 full-width at half-maximum (fwhm) (John et al. 2022a) and the scan range was from m/z 290 to m/z 2,000. The ddMS2 was recorded with a resolution of 17,500 fwhm and loop count was set to 10. A stepped normalized collision energy of 25 was chosen and m/z 100 was selected as fixed first mass. Analysis of the detected peptides was performed using the Proteome Discoverer software. All possible HETE modifications at e.g., cysteine, glutamic acid and aspartic acid residues were added to the inclusion list. Detailed settings are listed in supplementary information (Protocol SI 1).

For more sensitive and selective detection of identified modified peptides, the parallel reaction monitoring (PRM) mode was chosen as a second targeted approach and chromatography was carried out on an Acquity HSS T3 column (50 × 1 mm I.D. 1.8 μm, Waters) with solvent A and solvent B in gradient mode (30 μL/min, 40 °C, t[min]/B[%]: 0/2; 2.5/10; 3/20; 15/45; 16/98; 18/98; 19/2; 20/2, 10 min equilibration under starting conditions). Spray voltage was optimized to 3.0 kV. Eluates from tR 0 min to 20 min were directed toward the mass spectrometer. First, full scan MS (resolution 70,000 fwhm, scan range m/z 100 to m/z 1,500) was carried out followed by PRM scans. Resolution was set to 17,500 fwhm and fixed first mass was m/z 100. PRM analyses were carried out for: GAKPC192(-HETE)K [M + 2H]2+ (m/z 354.18258); KGAKPC192(-HETE)KSNK [M + 2H]2+ (m/z 582.81502); WGC199(-HETE)FPIHQAAFSGSK M + 3H]3+ (m/z 580.60593); EC213(-HETE)MEIILR [M + 2H]2+ (m/z 555.77143); EC213(-HETE)MEIILR [M + 3H]3+ (m/z 370.85005); ID339(-HETE)SEGR [M + 2H]2+ and IDSE341(-HETE)GR [M + 2H]2+ (m/z 390.68145); INTC462(-HETE)QR [M + 2H]2+ (m/z 419.69912); WDEC608(-HETE)LK [M + 2H]2+ (m/z 449.19588) and YLQC665(-HETE)PLEFTK [M + 2H]2+ (m/z 673.33017). The FreeStyle 1.3 software was used for data processing (Thermo Scientific).

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