2 5 dihydroxybenzoic acid

To analyze the mass of formed XG oligosaccharides, MALDI-TOF-MS (Bruker Daltonics, Billerica, Massachusetts, USA) was used as previously described [47 (link)]. The mass spectrometer was calibrated using maltodextrins (Avebe, Veendam, The Netherlands) in a mass range (m/z) of 500–3000 and a total of 300 spectra were collected for each measurement. Prior to analysis, samples were desalted using Dowex AG 50 W-X8 Resin (Bio-Rad Laboratories, Hempel Hempstead, UK). The desalted supernatants were dried under nitrogen and re-dissolved in water containing 20 mM LiCl to obtain lithium (Li)-adducts. 1 µL of each lithium-rich sample was mixed with 1 µL matrix solution (50% (v/v) acetonitrile in H₂O containing 12 mg/mL 2,5-dihydroxy-benzoic acid (Bruker Daltonics)) and dried under nitrogen.

The analysis of obtained extracts by Matrix-Associated-Laser-Desorption-Ionization with Time-of-Flight detector was performed as described previously [30 (link)]. A total of 1 µL of the extract was mixed with 1 µL of matrix solution (10 mg of 2.5-dihydroxybenzoic acid mL⁻¹ (Bruker Daltonics, Billerica, MA, USA) in acetonitrile/methanol/water (1:1:1, v/v/v) and 0.3 % trifluoroacetic acid (Sigma-Aldrich, St. Louis, MO, USA)). Then, 1 μL of the sample was directly spotted onto the target plate (MTP 384 plate ground steel BC, Bruker Daltonics, Billerica, MA, USA) and allowed to dry before analysis.
Measurements were performed in a reflector positive mode. A Peptide calibration standard II (Bruker Daltonics, Billerica, MA, USA) was used for calibration with m/z between 700 and 3500 Da. Mass spectra were compared with the available literature of peptaibols [28 (link),30 (link)]. Only very strong differences in the mass spectra (present versus not present) were considered relevant and used for the interpretation of results.

For matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), an Ultraflex workstation using FlexControl 3.3 (Bruker Daltonics) equipped with a nitrogen laser of 337 nm was used. The pulsed ion extraction was set on 80 ns. Ions were accelerated to a kinetic energy of 25 kV and detected in positive reflector mode with a set reflector voltage of 26 kV. The lowest laser energy required was used to obtain a good signal-to-noise ratio. A total of 200 spectra were collected for each measurement. The mass spectrometer was calibrated using a mixture of maltodextrins (Avebe, Veendam, The Netherlands) in a mass range (m/z) of 500–2,500. The peak spectra were processed by using FlexAnalysis software version 3.3 (Bruker Daltonics). Prior to analysis, samples were desalted by adding AG 50 W-X8 Resin (Bio-Rad Laboratories). To obtain lithium (Li) adducts, the supernatant was dried under nitrogen and re-suspended in 20 mM LiCl [28 (link)]. Each lithium-enriched sample of a volume of 1 µL was mixed with 1 µL of matrix solution (12 mg mL⁻¹ 2,5-dihydroxy-benzoic acid (Bruker Daltonics) in 30% (v/v) acetonitrile in H₂O), applied on an MTP 384 massive target plate (Bruker Daltonics) and dried under a stream of warm air.

HPLC methanol, acetonitrile, trifluoroacetic acid (TFA), formic acid, and ultrapure water (Milli-Q Millipore) were used throughout the study. Metronidazole (total impurities: ≤0.0005% phosphorus and ≤0.1% insoluble matter), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI-1640 and Dulbecco's Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich, St. Louis, MO, USA. Sephadex® LH20 was obtained from GE Healthcare Life Sciences. The matrix 2,5-dihydroxybenzoic acid (DHB) was purchased from Bruker Daltonics, and NaCl was obtained from Synth.

Methanol, potassium acetate, and ultrapure water were purchased from Wako Pure Chemical Industries (Osaka, Japan). Calibration standard peptides and 2,5-dihydroxybenzoic acid (DHB), a MALDI matrix, were purchased from Bruker Daltonics (Billerica, MA, USA). The antibiotic Bactramin was purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Minocycline hydrochloride was purchased from Sigma (St. Louis, MO, USA). All chemicals used in this study were of the highest purity available.

The 1-¹³C-D-glucose was purchased from Sigma-Aldrich^®. The matrices 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Bruker Daltonics.