ABSTRACT: Primary leaves of 10-day-old Phaseolus vulgaris (common bean) variety Black Valentine plants were infiltrated with 30 uM cFP, cGP, cLP, cWP, or auxin (indole-3-acetic acid, Sigma Aldrich), or water (control) using a flat-nosed 1 mL syringe. The two primary leaves on each plant were each infiltrated 8 times (4 times on each half leaf) with a single compound. Infiltration was performed on the abaxial side without puncturing the leaf, and each infiltration produced a 1 cm2 water-soaked area. After 24 hours, the leaf tissue at the site of infiltration was sampled with a 1 cm diameter cork-borer. Ten to twelve sites were collected from each plant to reach approximately 170 mg total tissue which was extracted in 1 mL 100% methanol. Four separate plants of each treatment were used to generate four replicates. Tissues were pulverized with 0.5 mm glass beads with a Qiagen PowerLyzer 24 bead mill (Qiagen, Hilden, Germany) 10 times at 5,000 rpm for 20 sec (cooled on ice for 2 min between cycles). Two matrix blank samples were prepared the same way but without tissue or compounds. The milled extracts were centrifuged for 20 min at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 uL 50% methanol/0.1% formic acid. Fifteen uL of each sample was pooled to create a quality control (QC) sample. Five uL of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-min linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per min. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Five subsequent injections of the QC were performed to generate tandem mass spectra (MS2). After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank 1, QC 1, sample replicate set 1, Blank 2, QC 2, sample replicate set 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. The samples also were analyzed in negative ion mode at -2,500 V but with the same other settings.
Bret Cooper 
