Exactive mass spectrometer

For lipid extraction, the lipophilic phase (see extraction protocol above) was collected and vacuum-dried. Samples were processed using ultra-performance liquid chromatography coupled with Fourier transform mass spectrometry (UPLC-FT-MS, Hummel et al., 2011 (link)), on a C8 reverse phase column coupled with an Exactive mass spectrometer (Thermo-Fisher)² in positive and negative ionization mode. Processing of chromatograms, peak detection and integration were performed using REFINER MS^® 6.0 (GeneData)³. Processing of mass spectrometry data included the removal of the fragmentation information, isotopic peaks, as well as chemical noise. Obtained features (m/z at a certain retention time) were queried against an in-house lipid database for further annotation.

Phytohormones were extracted and analysed according to [41 (link)]. Briefly, ~100 mg of frozen tissue from the same batches used for the RNA-Seq experiment were extracted twice with 1 ml of methanol/water 80 %, centrifuged at 20,000 g for 15 min. at 4 °C, the supernatant was passed through a C18 cartridge, and the samples were collected in a 5-ml tube for speed-Vac evaporation to dryness. The residue was resuspended in 1 ml methanol/water 20 %. Ten μl of filtrated extract were injected in a U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific) using a heated electrospray ionization interface. Mass spectra were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific). For quantification of the plant hormones, calibration curves were constructed for each analysed component (1, 10, 50, and 100 μg l⁻¹) and corrected for 10 μg l⁻¹ deuterated internal standards. Recovery percentages ranged between 92 and 95 %.

Optical rotations were measured on a Jasco P-1030 polarimeter (JASCO, Tokyo, Japan). Infrared spectra were measured on a Jasco FT/IR 4100 (JASCO, Japan). NMR spectra were recorded on a JEOL ECX 500 (500 MHz) or a Bruker DRX (500 MHz) spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are denoted in δ (ppm) relative to residual solvent peaks as internal standard (CDCl₃, ¹H δ 7.24, ¹³C δ 77.0). ESI–MS spectra were recorded on a Thermo Scientific Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or a SHIMADZU LCMS-2020 spectrometer (Shimadzu, Kyoto, Japan). High performance liquid chromatography (HPLC) experiments were performed with a SHIMADZU HPLC system equipped with a LC-20AD intelligent pump. LC–MS experiments were performed with amaZon SL (Bruker Daltonics, Bremen, Germany). Cell density for cytotoxic and anti-microbial assay was recorded on Tecan infinite^® M200 plate reader (Tecan, Salzburg, Austria) at Drug Discovery Scientific Research and Education Center-Open Lab. facility, Faculty of Pharmaceutical Sciences, Hokkaido University. All reagents were used as supplied unless otherwise stated.

LC-MS/MS analysis of the pooled fractions described above was used to generate a reference database of peptide markers defined by accurate masses and elution times, i.e., AMT tag (described in the paragraph below and refs. [46] (link)–[48] (link)). The AMT tag database then served as a comprehensive “look up” table for the subsequent higher throughput LC-MS analyses described below. For the AMT tag database generation, each of the 24 fractions corresponding to the purine-replete and purine-starved cells were analyzed using a 4-column, custom-built, capillary LC system coupled online via an in-house manufactured electrospray ionization (ESI) interface to an LTQ-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) [134] (link). To identify the eluting peptides, the LTQ-Orbitrap instrument was operated in a data-dependent MS/MS mode for the top six abundant precursor ions in each full MS scan. Following the AMT tag database generation, LC-MS analyses with full MS scan (400–2,000 m/z range) were performed on the 45 unfractionated purine-replete and purine-starved peptide samples described above to generate quantitative data. For this, samples were analyzed in random order using the same chromatographic and electrospray conditions as described for the LC-MS/MS analyses except that the LC system was interfaced to an Exactive Mass Spectrometer (Thermo Scientific).

For isotope tracing, cells were cultured in a 3 mL M9 medium supplied with the labeled/unlabeled carbon sources described in the main text. For ¹³CO₂ labeling, the experiment was performed in a 10 L desiccator that was first purged twice of the contained ambient air with a vacuum pump and refilled with an atmosphere of 80% air and 20% ¹³CO₂ (Cambridge Isotope Laboratories Inc., MA USA). All cultures were inoculated to an OD₆₀₀ 0.02 and grown at 37 °C until the stationary phase. Then, ~10⁹ cells (1 mL of culture with OD₆₀₀ = 1) were pelleted, washed once with ddH₂O, and hydrolyzed in 1 mL hydrochloric acid (6 M) at 95 °C for a duration of 24 h. Subsequently, the acid was evaporated by heating at 95 °C and the hydrolyzed biomass was re-suspended in ddH₂O.
Hydrolyzed amino acids were separated using ultra-performance liquid chromatography (Acquity, Waters, Milford, MA, USA) using a C18-reversed-phase column (Waters, Eschborn, Germany)^{90 (link)}. Mass spectra were acquired using an Exactive mass spectrometer (ThermoScientific, Dreieich, Germany). Data analysis was performed using Xcalibur (ThermoScientific, Dreieich, Germany). Prior to analysis, amino-acid standards (Merck, Darmstadt, Germany) were analyzed under the same conditions in order to determine typical retention times.

SK-ChA-1 cells were seeded in 6-wells plates and cultured until confluence. Cells were treated using the PDT protocol as described in “PDT protocol” (n = 3 per group). After 90 min, the cells were washed with 1 mL cold PBS and the cells were lysed in 1 mL lysis buffer (40% acetonitrile, 40% methanol, 20% water). The cells were scraped and transferred to 2-mL centrifuge tubes that were shaken for 10 min at 4 °C. Next, the samples were centrifuged for 15 min at 20,000×g (4 °C), after which the supernatant was aspirated and stored at −80 °C. LC-MS analysis was performed on an Exactive mass spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 autosampler and pump (Thermo Scientific). The MS operated in polarity-switching mode with spray voltages of 4.5  and −3.5  kV. Metabolites were separated using a Sequant ZIC-pHILIC column [2.1 × 150 mm, 5 µm, guard column 2.1 × 20 mm, 5 µm (Merck)] using a linear gradient of acetonitrile and eluent A (20 mM (NH₄)₂CO₃, 0.1% NH₄OH in ULC/MS grade water [Biosolve, Valkenswaard, the Netherlands)]. The flow rate was set to 150 µL/min. Metabolites were identified and quantified using LCquan software (Thermo Scientific) on the basis of exact mass within 5 ppm and further validated in accordance with the retention times of standards. Peak intensities were normalized based on total ion count.