Quantification of Phytohormones via Mass Spectrometry

Phytohormones were quantified as described in Geuss et al. (2018) (link) and see Supplementary Data Sheet S1 and Supplementary Table S1 for details on mass spectrometry. In brief, 100–110 mg of fresh leaf material (n = 4 samples/population/treatment) were ground and extracted two times with 1.0 ml ethyl acetate. Deuterated internal standards were included for salicylic acid (SA; OlChemIm Ltd., Olomouc, Czechia), abscisic acid (ABA; OlChemIm Ltd.), jasmonic acid (JA; Purity Compounds Standards GmbH, Cunnersdorf, Germany) and jasmonic acid-isoleucine (JA-Ile; HPC Standards GmbH). Extracts were vacuum-dried and re-eluted in 0.4 ml 70% MeOH containing 0.1% formic acid (v/v). Phytohormones were separated on a UPLC C18 column in a water^® and MeOH gradient (ACQUITY UPLC BEH-C18, 50 × 2.1 mm, particle size 1.7 μm), fragmented and detected in an ESI-MS/MS-qTOF detector (Synapt G2-S HDMS; Waters^®, Milford, MA, United States) and quantified according to the respective internal standard.

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Calf O.W., Lortzing T., Weinhold A., Poeschl Y., Peters J.L., Huber H., Steppuhn A, & van Dam N.M. (2020). Slug Feeding Triggers Dynamic Metabolomic and Transcriptomic Responses Leading to Induced Resistance in Solanum dulcamara. Frontiers in Plant Science, 11, 803.

Publication 2020

ACQUITY UPLC BEH-C18 Synapt G2-S HDMS

Abscisic acid Ethyl acetate Formic acid Isoleucine Ja ile Jasmonic acid Leaf Mass spectrometry Ms ms Phytohormones Salicylic acid Samples treatment Vacuum

Corresponding Organization : Friedrich Schiller University Jena

Other organizations : Freie Universität Berlin, University of Hohenheim, Martin Luther University Halle-Wittenberg

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Variable analysis

independent variables

Leaf material samples (100–110 mg of fresh leaf material)

dependent variables

Phytohormone levels (salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and jasmonic acid-isoleucine (JA-Ile))

control variables

Number of samples per population/treatment (n = 4)
Extraction method (two-time extraction with 1.0 ml ethyl acetate)
Internal standards (deuterated standards for SA, ABA, JA, and JA-Ile)
Separation method (UPLC C18 column with water and MeOH gradient)
Mass spectrometry method (ESI-MS/MS-qTOF detection)

positive controls

Deuterated internal standards for phytohormones

negative controls

Not explicitly mentioned

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