| Description
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Sample Preparation
Thirty 1-week-old plants were frozen in liquid N2 and ground to powder, which was used for methanol extraction. The extracts were filtered through disposable membrane filter units (DISMIC-13JP, ADVANTEC), evaporated under N2 atmosphere, and stored at −80°C until use. Upon FT-ICR/MS analysis, the extracts were dissolved in 50% (v/v) acetonitrile/water. IMCs for the positive ion mode were lidocaine ([M + H]+ = 235.18049), prochloraz ([M + H]+ = 376.03809), reserpine ([M + H]+ = 609.28066), and bombesin ([M + 2H]2+ = 810.41481). A set of 2,4-dichlorophenoxy acetic acid ([M−H]− = 218.96212), ampicillin ([M−H]− = 348.10235), 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid ([M−H]− = 613.38920), and tetra-N-acetylchitotetraose ([M−H]− = 829.32078) were used as the IMCs in the negative ion mode analysis.
FT-ICR/MS Conditions
Mass analysis was done using an IonSpec Explorer FT-ICR/MS (IonSpec) equipped with a 7-tesla actively shielded superconducting magnet. Ions were generated from an ESI source with a fused silica needle of 0.005-inch i.d. Samples were infused using a Harvard syringe pump model 22 at a flow rate of 0.5 to 1.0 μL min−1 through a 100-μL Hamilton syringe. All the experimental events were controlled using Omega8 software (IonSpec). Briefly, the potentials on the electrospray emitters were set to 3.0 kV and −3.0 kV for the positive and the negative electrosprays, respectively. The base pressure in the source region was approximately 5 × 10−5 torr (1 torr = 133.3 Pa). For the positive and negative electrosprays, sample solutions were prepared in 50% (v/v) acetonitrile/water with 0.1% (v/v) of formic acid and ammonium hydroxide, respectively. Ionized metabolites were accumulated for a period of 2,500 to 5,000 ms in a hexapole ion trap/guide and transferred through a radiofrequency-only quadrupole into the FT-ICR cell in the superconducting magnetic field, where they were again trapped. The direct current potentials in the positive and negative ion mode analyses were −2 V and 2 V during the ion accumulation and 2 V and −2 V for the ion transfer into the FT-ICR cell, respectively. These ions trapped in the hexapole were extracted for the transfer into the FT-ICR cell. In the positive and negative ion modes, the potentials on the extraction plate were 12 V and −12 V during the ion trapping and were reversed to −2 V and 2 V for the extraction. The base pressure in the analyzer region was set to the level of approximately 4 × 10−10 torr. ESI-MS spectra were acquired over the m/z range 55 to 1,000 from 1,024,000 independent data points. MS/MS analyses were done using the sustained off-resonance irradiation SORI-CID methods (Gauthier et al., 1991; Laskin and Futrell, 2005). SORI Rf was set at 0.5 to 1.5 V, and the N2 collision gas was used with 400-ms pulse.
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