Branched-chain amino acid catabolism promotes ovarian cancer cell proliferation via phosphorylation of mTOR

ABSTRACT: Slides were placed in a humidified incubator at 37C and 5% CO2 and cocultured for 4 days. After 4 days, the 8-well chamber was detached from the glass slide, and omental tissue was removed with a razor blade prior to desiccation. Agarose cocultures were desiccated on the ITO-glass slide in an incubator at 30C on a home-built spinning apparatus for 4 hours. (Lusk JASMS 2022) MALDI matrices alpha-cyano-4-hydroxycinnamic acid (CHCA (98%), C2020, Sigma-Adlrich) and 2,5-dihydroxybenzoic acid (DHB (98%), 149357, Sigma-Aldrich) were recrystallized in-house as previously described. The MALDI matrix used for MSI was a 50:50 mixture of CHCA:DHB at 10 mg/mL dissolved in 90:10 ACN:H2O with 0.1% TFA applied using a TM sprayer (HTX Imaging). Before MSI analysis, slides were scanned using Tissue Scout (Bruker Daltonics) for the initial screen and Epson Perfection V850 Pro for subsequent experiments. Scanned images were used to guide irradiation. MSI data was acquired using timsControl v2.0.51.0_9669_1571 and flexImaging 5.1 software for the initial screen, fleximaging 7.4 for subsequent experiments at 100 micron spatial resolution on a timsTOF fleX mass spectrometer (Bruker Daltonics). Data were collected using a mass range of 50-1500 Da in positive ion mode with the laser width set to 100 micron imaging and the laser power set to 90%. At each raster point, 1,000 laser shots were delivered at a frequency of 1,000 Hz. The instrument was calibrated manually using phosphorus red prior to imaging. MSI data was analyzed, and statistical analysis was performed using SCiLS Lab version 2023c core (Bruker Daltonics). All spectra were normalized to the root mean square (RMS).

INSTRUMENT(S): timsTOF fleX

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Laura Sanchez  

PROVIDER: MSV000095459 | MassIVE | Fri Jul 26 15:17:00 BST 2024

REPOSITORIES: MassIVE