Low concentration tuning mix

Ten microliters of freeze-dried samples dissolved in 70% EtOH (1 mg/mL) were injected into an ultra-performance liquid chromatography–time of flight mass spectrometry (UPLC-TOF-MS) system, which consisted of an Acquity™ UPLC (Waters, Milford, MA) and a MicroQTOF II mass spectrometer (Bremen, Germany). UPLC separation was performed with an Acquity UPLC™ BEH C18 column (1.7 μm, 50 mm × 2.1 mm i.d., Waters) at 40°C, using solvent A (0.1% formic acid in water) and solvent B (MeCN containing 0.1% formic acid).
Samples were eluted from the column using a linear gradient of 5% solvent B from 0.2 min to 90% solvent B at 13.16 min. The flow rate of the mobile phase was 0.4 mL/min. The TOF-MS was operated in the positive ion mode using an electrospray ionization source (ESI+). The detector conditions were as follows: capillary voltage at 4500 V, nebulizer at 1.8 bar, drying gas flow at 10 L/min, drying gas temperature 200°C, and the mass range between 50 and 2500 m/z. All analyses were performed using a low-concentration tuning mix (Agilent Technologies, Palo Alto, CA) to calibrate accurate mass. MS peak data from UPLC-TOF-MS analyses were subjected to Signpost™ software (Reifycs, Tokyo) for peak detection and integration.

MTBE, dichloromethane, and ammonium formate were from Sigma-Aldrich (Saint Louis, MO, USA). Ammonia solution in water (25%) was obtained from Merck KGaA (Darmstadt, Germany). MS additive formic acid (98%) was purchased from Chem-Lab NV (Zedelgem, Belgium). MS-grade isopropanol and acetonitrile were obtained from J. T. Baker (Phillipsburg, NJ, USA), and methanol was from VWR Chemicals (Darmstadt, Germany). Ultrapure water was produced in-house by Direct- Q^® 3UV (Millipore, Vienna, Austria). For MS calibration, a low concentration tuning mix was purchased from Agilent Technologies (Santa Clara, CA, USA).

Metabolites in the colorectal adenoma were imaged using a timsTOF fleX mass spectrometer (Bruker Daltonics, Billerica, MA) operated in negative ion mode in full scan mode for m/z 50–600. The instrument was tuned using a low concentration tuning mix (Agilent Technologies, Santa Clara, CA) injected into the ESI source. MALDI MSI was performed at 10 µm spatial resolution. The frequency of the laser was set to 10,000 Hz, with 1000 laser shots per 10 µm pixel. The data were visualized using SCiLS Lab software (version 2021c premium, Bruker Daltonics, Billerica, MA) with total ion count normalization. Fold change measurements were determined from clusters of pPDH+/pPDH− cells and their ion intensities for hexose-phosphate, pentose phosphate, succinate, fumarate, alpha-ketoglutarate, palmitoleic acid, and oleic acid. In each image, ten ROIs (Region of Interest) consisting of ten pixels each were selected for the analysis based on palmitoleic acid ion distribution as a positive marker for pPDH+ cell clusters, and the analysis was performed from both pPDH+ and pPDH− cell clusters. We analyzed three independent sections acquired from an APCmin/SIRT6^fl/fl/VillinCre adenoma (ten ROIs per section).