The purified peptidic samples were pre-concentrated on a pepmap LC trapping column (Thermo Scientific, Waltham, MA, USA, 0.3 × 5 mm) at a rate of 5 μL of Buffer A (0.1% Formic acid in water) for 5 min. Then the samples were injected onto a 50 cm long pepmap column (Thermo Scientific, Waltham, MA, USA) placed in an oven set to 55 °C, using a gradient of 100% Buffer A (0.1% Formic acid in water) to 27.5% Buffer B (0.1% Formic acid in acetonitrile) in 35 min followed by an increase to 40% buffer B in 2.5 min and a second increase to 99% Buffer B in 0.5 min and then kept constant for 1 min. Finally, the column was equilibrated for 15 min prior to the subsequent injection. A full MS was acquired using a Q Exactive HF-X Hybrid Quadropole-Orbitrap mass spectrometer (Thermo Fischer, Waltham, MA, USA) in the scan range of 350–1500 m/z using 60 K resolving power with an AGC of 3 × 106 and max IT of 45 ms, followed by MS/MS scans of the 12 most abundant ions, using 15K resolving power with an AGC of 1 × 105 and max IT of 22 ms and an NCE of 28.
Q exactive hf x hybrid quadropole orbitrap mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer is an analytical instrument designed for high-resolution, accurate mass measurements. It combines a quadrupole mass filter and an Orbitrap mass analyzer to provide high-performance mass spectrometry capabilities.
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Lab products found in correlation
2 protocols using q exactive hf x hybrid quadropole orbitrap mass spectrometer
1
Peptide LC-MS/MS Workflow
2
Proteome Analysis by DIA-MS
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Samples (~1 mg) were separated on a PepMap RSLC C18 analytical column (75 mm  50 cm, 2 mm, 100 Å, nanoViper, Thermo Scientific) using the EASY-nLC™ 1200 liquid chromatography system (Thermo Scientific) coupled in-line with a Q Exactive HF-X Hybrid Quadropole-Orbitrap mass spectrometer (Thermo Scientific). Separation was achieved by running constant flow rate of 350 nL/min in 0.1% formic acid/99.9% water and a 120 min gradient from 10% to 90% (10e30% for 90min, 30e45% for 20min, 45e90% for 1min, 90% for 10min) elution buffer (80% acetonitrile, 0.1% formic acid, 19.9% water). A DIA operated under Xcalibur 4.1.31.9 was applied to record the spectra. The full scan MS spectra (340e1400 m/z) were acquired with a resolution of 60,000 after accumulation to a target value of 3e6 and a maximum injection time of 100 ms. The MS/MS data was recorded by 32 full fragmentation scans, using an isolation window adjusted to the number of precursors (Suppl. 5). The precursor ions within each isolation window were fragmented using HCD with a resolution of 30,000 applying an AGC of 1e6 and a maximum injection time of 120 ms.
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