Q exactive hf hybrid quadrupole orbitrap

Digests were loaded onto Evotips (Evosep, Odense, Denmark) according to the manufacturer’s instructions (Bache et al., 2018 (link)). Evotips were soaked in isopropanol and sequentially washed with 20 μl acetonitrile with 0.1% formic acid, equilibrated with 20 μl water with 0.1% formic acid (solvent A), loaded with 20 μl sample, washed with 20 μl solvent A and loaded with 100 μl solvent A. Tips were centrifuged for 60 s at 700 × g between each loading step. The Evotips were submerged in 0.1% formic acid on a 96-wells plate during sample loading to prevent drying. After the last step, tips were centrifuged for 20 s at 700 × g. LC was performed by the Evosep One (Evosep, Odense, Denmark) using the manufactures separation method of 11.5 min (100 samples/day) (Bache et al., 2018 (link)). The Evosep One was coupled to an Orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific, Bremen, Germany). The Q Exactive HF system was operated in PRM mode using the following parameters: a quadrupole isolation window of 0.6 m/z units, an automatic gain control target value of 1 × 10⁶ ions, a maximum fill time of 150 ms and a resolving power of 60,000 at 200 m/z. A normalized collision energy of 27% was used for all peptides. A retention time window of 2 min was used for each peptide.

Stored digests were thawed and loaded onto Evotips (Evosep, Odense, Denmark) according to the manufacturer’s instructions and as previously described (Bache et al., 2018 (link); Foudraine et al., 2019 (link)). LC-MS/MS was performed using the Evosep One (Evosep, Odense, Denmark) coupled to an Orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific, Bremen, Germany). LC was performed using the manufacturer’s separation method of 11.5 min (100 samples/day) (Bache et al., 2018 (link)). The Q Exactive HF system was operated in parallel reaction monitoring (PRM) mode. The following settings were used: a quadrupole isolation window of 0.6 m/z units, an automatic gain control target value of 1 × 10⁶ ions, a maximum fill time of 150 ms and a resolving power of 30,000 at 400 m/z. A normalized collision energy of 27% was used for all peptides. Measurements were unscheduled in the peptide selection experiments. Measurements were scheduled with retention windows of 2 min for each peptide in the subsequent experiments. LC-MS/MS data was analyzed in Skyline daily 19.1 or later (MacCoss Lab Software, University of Washington, United States). The mass spectrometry proteomics data have been deposited to the n ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019 (link)) partner repository with the dataset identifier PXD025363.

MALDI MSI measurements were performed on a QExactive™ HF Hybrid-Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany) coupled to an AP-SMALDI5 source (TransMIT GmbH, Gießen, Germany) equipped with a λ = 343 nm solid state laser operating at a repetition rate of 100 Hz. Measurements were carried out in positive ion mode with a mass range of m/z 600–1000 with one scanning event per pixel at a mass resolution of 240 k @ m/z 200 full width at half maximum (FWHM). All measurements were performed with a fixed C-trap injection time of 500 ms. Step sizes were set to 25 µm for the sagittal wild-type zebrafish line sections, 10 µm for D. magna sections and 5 µm for the coronal Casper-Tol-056 D. rerio line sections. Tentatively identification of lipids from D. rerio sections was based on online data base search and on tissue MS/MS of lipids with a precursor isolation window width of ± 0.2 m/z. Tentatively identification of lipids in D. magna sections was based on online data base search^{52 (link)}.

Metabolites were extracted from approximately 5 × 10⁶ cells (grown in cell culture dishes) by the addition of 500 µL of ice‐cold 80% aqueous methanol. Supernatants were combined and filtered using a 3‐kDa ultrafilter (Millipore), dried in a SpeedVac (Thermo Fisher Scientific), and subsequently stored at −80°C. On the day of analysis, the dried extracts were reconstituted in 60 μL of ice‐cold 80% aqueous methanol. A quality control sample was made by combining 5 µL of each sample. Sample analysis was performed using anion‐exchange chromatography (Thermo UltiMate 3000 UHPLC, Thermo Fisher Scientific) coupled directly to a high‐resolution Orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole‐Orbitrap, Thermo Fisher Scientific) as previously described 22 and detailed in online Supporting Information.

Peptides were re-suspended in 17 μl of 0.1 % TFA. A total of 5.0 μl were injected into a nano-HPLC device (Thermo Fisher Scientific) using a gradient from 4 % B to 90 % B (solvent A 0.1 % FA in water, solvent B 80 % ACN, 0.1 % FA in water) over 90 min at a flow rate of 300 nl/min in a C18 UHPCL column (Thermo Fisher Scientific). Data was acquired in PRM positive mode using a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ (Thermo Fisher Scientific) to identify and quantify specific N-terminal peptides of histone H3 and histone H4 proteins and their PTMs. MS1 spectra were acquired in the m/z range 250-1600 with resolution 60,000 at m/z 400 (AGC target of 3x10⁶). MS2 spectra were acquired with resolution 15,000 to a target value of 2x10⁵, maximum IT 60ms, isolation 2 window 0.7 m/z and fragmented at 27 % normalized collision energy. Typical mass spectrometric conditions were: spray voltage, 1.5kV, no sheath and auxiliary gas flow, heated capillary temperature, 250°C.

Samples were analyzed on an Ultimate 3000 RSLC system coupled to QExactive HF-X hybrid Quadrupole-Orbitrap, QExactive HF hybrid Quadrupole-Orbitrap, or QExactive Plus Hybrid Quadrupole-Orbitrap mass spectrometers (Thermo Fisher Scientific). Acquisition parameters were tuned for the individual experiments and the performance of the mass spectrometer, and can be found in the Source data file. For LFQ, correct peptide ions and peptide ions with amino acid substitutions were targeted in their highest populated charge state. Sets of interference-free fragment chromatograms were extracted using the Skyline software^{72 ,73 (link)} and abundance differences were estimated based on the sum of the individual integrated fragment intensities.