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Ulnaria_parasites_temperature_cell_profiling

ABSTRACT: Comparison of metabolic profiling from algal cell extracts using UHPLC-HRMS (Orbitrap Q Exactive). Benthic diatom Ulnaria were healthy or infected with either chytrid or oomycete parasites, and subjected to temperature increase. Data from 20231125_xx was analyzed using C18 column, data from 20251125_xx_zh was analyzed using Zic-Hilic column; both dataset consists of cell profiling with polarity switch (MS1) and ddMS acquisition on the QC pool sample in positive and negative polarity (indicated by neg_ID or pos_ID in the title name). QC pool blank was also run with the polarity switch to acquire MS1 data. Data from 20241222_xx was analyzed using RP18 column, in negative polarity and using ddMS acquisition for all samples, including blank and QC pool sample. In supplementary files are the study and result files from Compound Discoverer, the metadata table with normalization factor, sample code and condition, and the resulting peak lists after peak deconvolution that were used for annotation (.xls) and for statistical analysis in MetaboAnalyst (.csv)

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Ulnaria (ncbitaxon:426668)

SUBMITTER: Dr. Marine Vallet

PROVIDER: MSV000097103 | MassIVE | Tue Feb 11 23:50:00 GMT 2025

REPOSITORIES: MassIVE

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Project description:P. savastanoi pv. phaseolicola isolate R5 was cultured in 10 mL Luria broth with 400 uM genistein or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform. Replicate R5 cultures of each treatment were centrifuged at 5,000 x g for 10 min., washed once in 1 mL phosphate buffered saline, centrifuged at 5,000 x g for 10 min., resuspended in 1 mL of 1:1:1:1 acetone/acetonitrile/methanol/water and 1,2500 pmol prednisone, and pulverized in the bead mill as before. Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without genistein). The milled extracts were centrifuged for 20 minutes at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 uL 50% methanol/0.1% formic acid. Fifteen uL of each sample was pooled to create a quality control (QC) sample. Five uL of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Three subsequent injections of the QC were performed to generate MS2 spectra. After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank 1, QC 1, sample replicate set 1, Blank 2, QC 2, sample replicate set 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. The samples also were analyzed in negative ion mode at -2,500 V but with the same other settings.

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Ulnaria_parasites_temperature_cell_profiling

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