Hybrid quadrupole orbitrap mass spectrometer

Different myosin samples were digested through the same procedure as described above. Then, the myosin samples were heated at 100 °C for 5 min to stop the enzymatic reaction. The digested products were identified based on the method of our previous study [8 (link)], with slight modifications. Briefly, the myosin mixtures were centrifuged at 14,000× g for 20 min to obtain the pure peptide. Subsequently, these peptides were separated by two different C18 columns (2 cm × 200 μm, 5 μm; 75 μm × 100 mm, 3 μm). In this sense, buffers A (0.1% formic acid, water) and B (0.1% formic acid, 84% acetonitrile) were used in the separation. Peptides were then identified through a hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Tolerance: 10 ppm, variable modification: Met oxidation. Proteome Discover-1.4 was applied to match the myosin with peptide data (http://www.uniprot.org/, accessed on 1 December 2022 ).

Samples were analyzed by HPLC-MS/MS using a Dionex UltiMate 3000 system coupled with a Q Exactive^TM Focus, high resolution and high mass accuracy, hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using a Supelco Ascentis C18 column (2.1 × 150 mm, 3 µm). Linear gradient elution (0 min 2% B, 1 min 2% B, 11 min 90% B, 11.5 min 90% B, 12 min 2% B, 15 min 2% B) with eluent A (0.1% HCOOH in water, vol/vol%) and eluent B (0.1% HCOOH in acetonitrile/water, 80:20, vol/vol%) was used at a flow rate of 0.2 mL/min at 40 °C. Detection of daunomycin was in ESI + mode using Parallel Reaction Monitoring (PRM) at a resolution of 17,500 FWHM. The precursor ion (m/z: 528.19) was selected for analysis. The isolation window with was set to 2 m/z. Normalized collision energy (NCE) was 15%. LC-MS/MS data were visualized and analyzed by Xcalibur^TM software (Thermo Fisher Scientific). Peak area from the Extracted Ion Chromatograms (EIC) of the ion transition m/z 528.19 -> 321.07 (±0.5 Da) were used to calculate relative daunomycin concentration.