The acylsugar extraction interactive protocol is available in Protocols.io at http://dx.doi.org/10.17504/protocols.io.xj2fkqe. Leaf surface acylsugars were extracted from single leaflets with 1 ml of a mixture of isopropanol (J.T.Baker, Phillipsburg, NJ)/acetonitrile (Sigma-Aldrich, St. Louis, MO)/water (3:3:2) with 0.1% formic acid and 1 μM telmisartan (Sigma-Aldrich, St. Louis, MO) as a high-performance liquid chromatography (HPLC) standard. The leaf tissue was gently agitated on a rocker in this extraction solvent for 2 min. The extraction solvent was collected and stored in 2-ml LC-MS vials at −80°C.

LC-MS samples (both enzyme assays and plant samples) were run on a Waters ACQUITY UPLC coupled to a Waters Xevo G2-XS QToF mass spectrometer. Ten microliters of the acylsugar extracts were injected into an Ascentis Express C18 HPLC column (10 cm by 2.1 mm, 2.7 μm) (Sigma-Aldrich, St. Louis, MO), which was maintained at 40°C. The LC-MS methods used the following solvents: 10 mM ammonium formate (pH 2.8) as solvent A and 100% acetonitrile as solvent B. Compounds were eluted using one of two gradients.

A 7-min linear elution gradient consisted of 5% B at 0 min, 60% B at 1 min, 100% B at 5 min, held at 100% B until 6 min, 5% B at 6.01 min, and held at 5% B until 7 min. A 21-min linear elution gradient consisted of 5% B at 0 min, 60% B at 3 min, 100% B at 15 min, held at 100% B until 18 min, 5% B at 18.01 min, and held at 5% B until 21 min.

The MS settings were as follows for negative ion-mode electrospray ionization (ESI−): capillary voltage, 2.00 kV; source temperature, 100°C; desolvation temperature, 350°C; desolvation nitrogen gas flow rate, 600 liters/hour; cone voltage, 35 V; and mass range, m/z 50 to 1000 (with spectra accumulated at 0.1 s per function). Three separate acquisition functions were set up to test different collision energies (0, 15, and 35 V).

The MS settings for positive ion-mode ESI were as follows: capillary voltage, 3.00 kV; source temperature, 100°C; desolvation temperature, 350°C; desolvation nitrogen gas flow rate, 600 liters/hour; cone voltage, 35 V; and mass range, m/z 50 to 1000 (with spectra accumulated at 0.1 s per function). Three separate acquisition functions were set up to test different collision energies (0, 15, and 45 V). Lock mass correction was performed using leucine enkephalin as the reference compound for data acquired in both negative and positive ion mode.

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