5975c msd single quadrupole mass spectrometer

All samples were analysed by normal flash pyrolysis GC/MS. The samples were pyrolysed using a CDS (Chemical Data Systems) 5200 series pyroprobe pyrolysis unit by heating at 600 °C for 20 s to fragment macromolecular components. These fragments were then analysed using an Agilent 7890A gas chromatograph fitted with a HP-5 fused column (J&W Scientific; 5% diphenyl-dimethylpolysiloxane; 30 m, 0.32 mm i.d., 0.25 μm film thickness) coupled to an Agilent 5975C MSD single quadrupole mass spectrometer operated in electron ionization (EI) mode (scanning a range of m/z 50–650 at 1 scan s⁻¹ with a 1 minute solvent delay; ionization energy 70 eV). The pyrolysis transfer line and injector port temperatures were set at 350 °C, the heated interface at 280 °C, the EI source at 230 °C and the MS quadrupole at 150 °C. Helium was used as the carrier gas and the samples were introduced in split mode in a ratio of 5:1. The oven was programmed from 40 °C (held for 5 minutes) to 250 °C at 4 °C min⁻¹, then to 320 °C at 20 °C min⁻¹, were it was kept for 5 min. Compounds were identified by comparison with spectra from the literature.

Cell extract of galacturonate-grown L. suebicus LCV1 (5.1 ± 0.4 g L^–1) was incubated for 30 or 180 min at 30°C in a reaction mixture containing triethanolamine (TEA) buffer (100 mM, pH 7.6), 2.5 mM MgCl₂.6H₂O, 5 mM ATP, and 5 mM mannonate or no ATP and with 5 mM 6-phosphogluconate, unless stated otherwise. Samples were centrifuged (13,000 × g, Microfuge, ThermoFisher Scientific, Waltham, NC, United States) after incubation and the supernatant was collected; 100 μL of the supernatant was frozen at −80°C (U101 Innova freezer, Eppendorf, Hamburg, Germany) and freeze-dried over-night (Mini Lyotrap freeze-dryer, LTE Scientific Ltd., Greenfield, Oldham, United Kingdom). The supernatant was derivatized according to Niedenführ et al. (2016) (link) without addition of AAL-mix and analyzed using a 7890A gas chromatography system (Agilent, Santa Clara, CA, United States) coupled to a 5975C MSD single quadrupole mass spectrometer (Agilent, Santa Clara, CA, United States) according to de Jonge et al. (2011) (link); split ratio of 1: 50 for standards, split ratio of 1: 10 for samples. Identification of the peaks was done via MassHunter Workstation Qualitative Analysis software (Agilent, version B06.00) and comparison to the NIST Standard Reference Database (version 2.0).

To obtain the enrichment of intracellular free amino acids during the ¹³C labelled experiments, the extracting solution was concentrated to 100 μL of supernatant before being transferred to a glass vial. After lyophilization, 80 μL acetonitrile and 80 μL of N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA, Thermo Scientific) were added and the vial was incubated for 2 h at 60 °C. After that, the sample was centrifuged (10,000g, 2 min) and 160 μL of the supernatant was transferred to a GC glass vial with an insert. The sample was then analyzed by GC–MS instrument coupled to a 5975 C MSD single quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA).