Q exactive hf mass spectrometer

FOC4 was cultured on a PDA plate for 5 days. The edge hyphae were picked into 100 mL of PDB liquid medium and cultured until the concentration of spores reached 10⁶/mL. Then, the FOC4 spore suspension was inoculated into PDB liquid medium (1 L) and cultured for 2 days. Subsequently, FOC4 fermentation liquid continued to be co-cultivated under inducing by hung sterilized banana seedlings for 2 days. The culture supernatant was centrifuged at 3500 g for 30 min at 4 °C and filtered with a 0.45 μm filter (Millipore, Suzhou, China). Total proteins in the supernatant were extracted with a trichloroacetic acid (TCA)-acetone precipitation method and dissolved in protein lysate buffer (8 M urea, 100 mM Tris-HCl, pH 8.0, 1 mM PMSF protease inhibitor) [56 (link)]. All proteins were analyzed with the high-performance liquid chromatography (HPLC) liquid phase system Ultimate 3000 (Thermo Scientific, Waltham, MA, USA); mass spectrometry was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific, Waltham, MA, USA). The RAW files were identified and quantified using Mascot and Proteome Discoverer, and the results of filtration parameters were set as PSM FDR ≤0.01, Protein FDR ≤0.01 [57 (link)]. Label-free analysis of F. oxysporum was completed by Beijing Bio-Fly Bioscience Co., Ltd.

Pellet samples were excised as whole lanes from gels, reduced with TCEP, alkylated with iodoacetamide, and digested with trypsin. Tryptic digests were desalted by loading onto a MonoCap C18 Trap Column (GL Sciences), flushed for 5 min at 6 μL/min using 100% Buffer A (H₂0, 0.1% formic acid), then analyzed via LC (Waters NanoAcquity) gradient using Buffer A and Buffer B (acetonitrile, 0.1% formic acid) (initial 5% B; 75 min 30% B; 80 min 80% B; 90.5–105 min 5% B) on the Thermo Q Exactive HF mass spectrometer. Data were acquired in data-dependent mode. Analysis was performed with the following settings: MS1 60K resolution, AGC target 3e6, max inject time 50 ms; MS2 Top N = 20 15K resolution, AGC target 5e4, max inject time 50 ms, isolation window = 1.5 m/z, normalized collision energy 28%. LC-MS/MS data were searched with full tryptic specificity against the UniProt human database using MaxQuant 1.6.8.0. MS data were also searched for common protein N-terminal acetylation and methionine oxidation. Protein and peptide false discovery rate was set at 1%. LFQ intensity was calculated using the MaxLFQ algorithm (Cox et al., 2014 (link)). Fold enrichment was calculated as LFQ intensity from the ΔCLPB pellet divided by the LFQ intensity from the wild-type pellet. High confidence hits were quantified as minimum absolute fold change of 2 and p-value<0.05.

The dried TMT-labeled phosphopeptides were prefractionated using offline HILIC HPLC prior to being analyzed by mass-spectrometry as described by Sims et al., 2022 (link). The LC-MS/MS was performed on an UltiMate 3000 RSLC nano chromatographic system coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). The chromatographic separation was achieved via a 35-cm-long 100 µm inner diameter column packed in-house with 3 µm C18 reversed-phase resin (Reprosil Pur C18AQ 3 μm). The Q-Exactive HF was operated in data-dependent mode with survey scans acquired in the Orbitrap mass analyzer over the range of 380–1800 m/z with a mass resolution of 120,000. MS/MS spectra were performed after selecting the top 7 most abundant +2, +3, or +4 ions and a precursor isolation window of 0.7 m/z. Selected ions were fragmented by higher-energy collisional dissociation (HCD) with normalized collision energies of 28, with fragment mass spectra acquired in the Orbitrap mass analyzer with a monitored first mass of 100 m/z, mass resolution of 15,000, AGC target set to 1 × 10⁵, and maximum injection time set to 100 ms. A dynamic exclusion window of 30 s was specified.

Thermal proteome profiling (TPP) over a temperature range was performed with minor modifications to previously described, using the ALL-PO cell line [20 (link)]. Online liquid chromatography tandem mass spectrometry was performed using a Dionex UltiMate™ 3000 RSLCnano System coupled to a Q-Exactive-HF mass spectrometer (Thermo Fisher Scientific) [20 (link)]. Analyses of the acquired mass spectrometry TPP data for the identification of the drug targets was performed as previously described (Supplementary Methods) [21 , 22 (link)]. Cellular thermal shift assay (CETSA) temperature range and dose response time course experiments using western blotting readout were used to validate target engagement (Supplementary Methods).

Peptides were separated on an EASY-nLC 1200 HPLC system (Thermo Fisher Scientific), coupled online to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Peptides were loaded in buffer A (0.1% formic acid) into a column (75 µm inner diameter, 50 cm length) in-house packed with ReproSil-Pur C18-AQ 1.9 µm resin (Dr. Maisch HPLC GmbH), and eluted over a 150-min linear gradient of 5%–30% buffer B (80% ACN, 0.1% formic acid) with a 250 nl/min flow rate. The Q Exactive HF was operated with the Xcalibur software (Thermo Scientific) in a data-dependent mode with a survey scan range of 300–1,650 m/z, a resolution of 60,000 at 200 m/z, a maximum injection time of 20 ms, and an AGC target of 3e6. Up to 10 most abundant ions with charge 2–5 were isolated with a 1.8 m/z isolation window and subjected to higher-energy collisional dissociation (HCD) fragmentation with a normalized collision energy of 27. MS/MS scans were acquired with a resolution of 15,000 at 200 m/z, a maximum injection time of 55 ms, and an AGC target of 1e5. Dynamic exclusion was set to 30 s to avoid repeated sequencing.

All samples described above were stored at −80 °C until sample preparation for proteomic analysis. The samples were macerated with a clean pestle and washed with 25 μL of Pierce RIPA buffer. Then, they were digested using the standard gel-aided sample preparation protocol as described previously (31 (link), 60 (link)). Peptide samples were analyzed on a liquid chromatography tandem mass spectrometry platform, a Q-Exactive HF mass spectrometer (Thermo), and processed as described previously (31 (link)).