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Experimental Design, Materials and Methods

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Experimental Design, Materials and Methods

Plant materials

Mangosteen fruit were obtained from Universiti Kebangsaan Malaysia (UKM) mangosteen plots (Bangi, Selangor, Malaysia) (GPS coordinate: 2.922662°E, 101.786690°N) [3]. Each fruit was first tagged at anthesis and its development observed. Seeds were sampled and pooled from at least six biological replicates (independent fruit) at six, eight, ten, twelve and fourteen weeks after anthesis and stored at -80°C until experiment.

Metabolite extraction

Pooled whole seeds were finely grounded and three replicates (0.1 g fresh weight per replicate) were used for metabolite extraction [4] at each stages of development. Samples were homogenized (in 1400 µL of methanol) and incubated (15 min, 70°C). The extract was mixed briskly with 1 volume of water and centrifuged at 2200 × g. The supernatant was vacuum dried for four to six hours then stored at – 80°C until analysis.

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Liquid Chromatography-Mass Spectrometry (LC-MS) protocol

LC-MS analysis was carried out using Dionex liquid chromatography platform (Ultimate 3000 UHPLC+, Thermo Scientific) coupled with MS sytem MicroTOF-QIII (Bruker, Germany) MS system [1, 5-6]. The LC parameters were set as follows; 1.0 µL injection volume, column temperature 60°C and 0.3 mL/min flow rate. The LC mobile phase consisted of 0.1 % formic acid in water (A) and mobile phase B (100 % acetonitrile). The elution run for 35 min with the gradient parameters: 0 to 5 % B in 2 min, 5 to 40 % B in 2 min and 40 to 95 % B in 16 min. The mixture was then held at 95 % B for 2 min before increasing to 100 % B in 0.1 min.

The mixture was held at 100 % B for 4 min and then decreased to 5 % B in 2 min. The column was reconditioned with initial gradient for 7 min. Mass spectrometry analysis was performed using MicroTOF-Q hybrid quadrupole time of flight mass spectrometer (Bruker, Germany), outfitted with an electrospray ion source (ESI) using the following parameters; nebulizer pressure: 1.2 bar, drying gas: 8 L/min at 200°C, capillary voltage: 4500 V, end plate off set: -500 V, funnel 1 RF: 200 Vpp, funnel 2 RF: 200 Vpp. Analysis were done in positive ionization mode with spectra scan range set from 50 to 1000 m/z range. All spectra recorded were in centroid mode.

Mass spectrometry data handling

Raw data from MS were imported into ProfileAnalysis version 2.1 (Bruker, Germany). For bucketing, the parameters for Find Molecular Features were manipulated (S/N threshold: 5, correlation coefficient: 0.7, minimum compound length was: 10, smoothing width: 2). Bucket was generated with the following settings: retention time range (0.1 minutes to 30.00 minutes), mass range (50 m/z to 1000 m/z), normalization (largest bucket value in analysis). Then the data were tabulated using Microsoft Excel 2016.

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