Oasis hlb column

For SSA extraction, a 2.5% (w/v) SSA solution with 1 μM crotonoyl CoA was prepared. An extraction solution of 200 μL was added to cell pellets on wet ice and mixed. The samples were then centrifuged at 18,000× g for 15 min. Following centrifugation, supernatants were removed and transferred to glass LC-MS vials for analysis.
For extraction with 10% (w/v) TCA, a solution containing 1 μM crotonoyl CoA was prepared. An extraction solution of 200 μL was added to cell pellets and samples were resuspended by gentle pipetting. Solid phase extraction was then performed according to published methods [16 (link),21 (link),22 (link)]. Briefly, Oasis HLB columns (Waters) were first conditioned with 1 mL methanol, then equilibrated with 1 mL H₂O. After equilibration, TCA-extracted samples were placed onto columns, washed with 1 mL H₂O, and eluted with 1 mL of 25 mM ammonium acetate in methanol. Samples were dried overnight at 4 °C in a Centrivap benchtop vacuum concentrator (Labconco; St. Louis, MO, USA). Evaporated samples were reconstituted with 2.5% SSA and moved to LC-MS vials for analysis.

A 1260 Infinity quaternary ultra high performance liquid chromatography (UHPLC) system (Agilent, Waldbronn, Germany) equipped with a binary pump, vacuum-chambered microdegasser, thermostatically controlled autosampler, and column compartment were used in the study. The system was also equipped with a diode-array detector and an Agilent 6540 UHD accurate-mass quadrupole time-of-flight (Q-TOF) mass spectrometer. Electrospray ionization (ESI) was applied in negative ion mode. A full-scan MS was recorded within the mass range m/z 400–1650 and then quadrupole precursor ion selection MS/MS was realized for m/z 50–1650. The following MS/MS parameters were applied: drying gas flow rate 11.0 L min⁻¹; shielding gas flow rate 10.0 L min⁻¹; nebulizer gas pressure 20 psi; skimmer voltage 60 V, octopole voltage 750 V; capillary voltage 4000 V; shielding/drying gas at 350/400 °C; fragmentor voltage 150 V. Nitrogen was used in the ion source and the collision cell. Data were collected using the Agilent Mass Hunter software, version B.04.01.
Sample preparation was performed using a CentriVap vacuum concentrator (Labconco, Kansas City, MO, USA), a model 5424 centrifuge (Eppendorf AG, Hamburg, Germany), a ThermoMixer (VWR, Randor, PA, USA), a laboratory pH meter (Elmetron CP-505, Zabrze, Poland), and Oasis^® HLB columns for solid-phase extraction (60 mg/3 ml; Waters, Milford, MA, USA).

HEK293T cells were transfected with 5 µg of DNA for each construct using the calcium phosphate transfection method. After 2 d, cells were harvested in ice-cold lysis buffer containing 8M urea, 50 mM ammonium bicarbonate, and Halt protease phosphatase inhibitor mixture (Pierce). Phosphopeptides were enriched as previously described (81 (link), 82 (link)), with slight modifications. Briefly, proteins were reduced using 10 mM DTT and subsequently alkylated using 20 mM iodoacetamide. Remaining iodoacetamide was quenched by adding additional DTT at a final concentration of 20 mM. Peptides were digested overnight (16 h) at room temperature with shaking (200 rpm) using trypsin (1:50 weight per weight as a protease). Peptides were desalted using Oasis HLB columns (Waters). After drying down in a SpeedVac (Christ), peptides were resuspended in 5% acetic acid. Phosphopeptides were enriched using Fe-NTA IMAC resin columns (Pierce). Eluted phosphopeptides were dried and were subsequently cleaned using C18 ZipTips. Eluted phosphopeptides were dried and resuspended in 0.1% formic acid for final analysis.

Quantification of plant hormones was conducted using reverse-phase ultra-high-performance liquid chromatography (UHPLC) coupled with electrospray ionization tandem triple quadrupole mass spectrometry (ESI/TQ MSMS) using multiple reaction monitoring as described previously (26 (link)). In brief, A. thaliana leaves were flash-frozen in liquid nitrogen and ground to powder. Samples (70 mg) were shaken vigorously in isopropanol:H₂O:HCl (2:1:0.002) for 1 h at 4 °C. Following extraction, dichloromethane was added, and samples were again shaken vigorously for an additional 30 min at 4 °C. Following centrifugation (3500 × g at 4 °C), the bottom layer was removed using a glass syringe. Samples were then dried under nitrogen, redissolved in 0.10 mL of MeOH and 1 mL of 1% acetic acid, purified over Oasis HLB columns (Waters), washed with 1% acetic acid, and eluted with 80% MeOH and 1% acetic acid. The eluted samples were then dried again under nitrogen and redissolved in 25 μL of MeOH and 25 μL of 1% acetic acid. A 10-μL volume of each sample was separated on a Waters Acquity UPLC BEH C18 column (2.1 × 150 mm; 1.7 μm) at 60 °C, using a Waters Acquity UHPLC system. A Waters Xevo TQ MSMS system was used to identify the different hormones, and absolute quantification was obtained using stable isotope label internal standards for JA and SA (26 (link)).