Q exactive hf spectrometer

Peptides were resuspended in 17 μl of 0.1% Trifluoroacetic Acid (TFA). A total of 5.0 μl were injected into a nano-HPLC device (Thermo Fisher Scientific, UltimateNano3000) using a gradient from 4% solvent B to 90% solvent B (solvent A 0.1% Formic Acid (FA) in water, solvent B 80% Acetonitrile (ACN), 0.1% FA in water) over 90 min at a flow rate of 300 nl min⁻¹ in a C18 Ultra-High Pressure Liquid chromatography (UHPLC) column (Thermo Fisher Scientific, 164534). Data were acquired in parallel-reaction monitoring (PRM)-positive mode using a Q Exactive HF spectrometer (Thermo Fisher Scientific) to identify and quantify specific N-terminal peptides of histone H3 and histone H4 proteins and their PTMs. One survey MS1 scan and nine MS2 acquisitions from the precursor m/z value in the inclusion list was performed. MS1 spectra were acquired in the m/z range 250–1,600 with a resolution of 30,000 at m/z 400 (AGC target of 3 × 10⁶). PRM spectra were acquired with resolution 15,000 to a target value of 2 × 10⁵, maximum injection time (IT) 60 ms, isolation 2 window 0.7 m/z and fragmented at 27% normalized collision energy. Typical mass spectrometric conditions were as follows: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250 °C. MS histone PTM analysis and quantification are detailed in the Supplementary Methods.

First, 100 µg of venom protein samples was placed in a centrifuge tube with 5 μL 1 M DTT and incubated at 37 °C for 1 h. Then, 20 μL indole-3-acetic acid (IAA) was added at room temperature and the tube was kept in the dark for 1 h. Samples were centrifuged and the supernatant was removed. After addition of 100 μL UA, samples were centrifuged and the supernatant was removed again, after which 100 μL 50 mM NH₄HCO₃, was added, followed by centrifugation and removal of the supernatant once more. Trypsin was added at a 50:1 protein/enzyme ratio, followed by hydrolysis at 37 ºC for more than 12 h for liquid chromatography with tandem mass spectrometry analysis.
Second, the samples were separated using high-pressure liquid chromatography (Ultimate 3000, Thermo Scientific, USA) with a C18 trap column (C18 1.9 m 0.15 × 120 mm). The gradient was composed of 0.1% methanoic acid (A) and 0.1% methanoic acid/80% acetonitrile (B), and the linear gradient was set as follows: 0–8 min for 94–91% A, 8–24 min for 91–86% A, 24–60 min for 86–68% A, 60–75 min for 68–32% A, and 75–80 min for 32–5% A. The flow rate was 600 nL.min^-1. Finally, each separated sample was analyzed with a Q-Exactive HF spectrometer (Thermo Scientific, USA); see Fig. S16 for parameters. The acquired datasets (.raw files) were analyzed using MaxQuant and the built-in Andromeda search engine against the UniProt database.