During this experiment, all fractions used to generate the DDA library were detected by Thermoscience QExactive HF X mass spectrometer. Next: take the peptide (1.5 μg). Load it onto EASY Trapcolumn (Thermo Scientific, P/N 164946, Waltham, MA, USA), and then separate the sample on the corresponding analytical column (ES802, 2 um, 75 um × 25 cm). Control the concentration gradient of buffer B to 250 nl/min, 120 min. During MS detection, the parameters set are as follows: scanning range 300–1800 m/z, resolution 60,000, AGC 3e6, and maximum IT 25 ms. After scanning ddMS2 for 20 times, start a complete MS–SIM scan. The parameters set during MS2 scanning are as follows: the resolution is 15,000, AGC is 5e4, and the maximum IT is 25 ms.
Q exactive hf x mass
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q-Exactive HF-X™ is a high-resolution, accurate-mass Orbitrap mass spectrometer. It is designed to provide high-quality data for a wide range of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Dionex ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Easy trap column, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap100 c18 3 μm, by Thermo Fisher Scientific (1 mentions)
Ziptip, by Merck Group (1 mentions)
Easy spray column, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Orbitrap ms, by Thermo Fisher Scientific (1 mentions)
Qe hfx mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (1 mentions)
6 protocols using q exactive hf x mass
1
Peptide Analysis Using QExactive HF X
2
Peptide Quantification by LC-MS/MS
3
Peptide Desalting and Mass Spectrometry
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Each sample of digested peptides was desalted with a ZipTip® with 0.6 μL resin bed volume (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The eluent was dried and resuspended in 0.1% formic acid, and was analyzed using a Q Exactive HFX mass spectrometer connected to an Easy nLC 1200 ultra high-pressure chromatography system (Thermo Scientific, Waltham, MA, USA). The samples were loaded onto a reverse-phase nano-trap column (75 μm interior diameter × 2 cm, Acclaim PepMap100 C18 3 μm, 100 Å, Thermo Fisher Scientific, Waltham, MA, USA) with mobile phase A (0.1% formic acid and 2% acetonitrile), and separated over an EASY-Spray column, (ES803A, 75 μm i.d. × 50 cm C18 2 μm, 100 Å, Thermo Fisher Scientific, Waltham, MA, USA) using a gradient (2% to 32% over 60 min) of mobile phase B (0.1% formic acid, 80% acetonitrile) at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 275 °C and a potential of 2100 V applied to the emitter. All the data were acquired with the mass spectrometer operating in automatic data dependent switching mode. A high-resolution (60,000) MS precursor ion scan (350–1500 m/z range) was performed to select the 10 most intense ions for the subsequent fragmentation and MS/MS analysis using HCD (NCE 29 at 15,000 resolution) each duty cycle.
4
UHPLC-MS/MS Analysis of Metabolites
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LC–MS/MS analysis was performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The analysis was conducted with the following elution gradient: 0 ~ 0.5 min, 95% B; 0.5 –7.0 min, 95% ~ 65% B; 7.0 –8.0 min, 65% ~ 40% B; 8.0 – 9.0 min, 40% B; 9.0 –9.1 min, 40% ~ 95% B; 9.1 –12.0 min, 95% B. The column temperature was maintained at 30 °C. The auto-sampler temperature was 4 °C, and the injection volume was 2 µL. The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra in the information-dependent acquisition (IDA) mode under the control of acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluated the full-scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate, 50 Arb; Aux gas flow rate, 10 Arb; capillary temperature, 320 °C; full MS resolution, 60,000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; spray voltage, 3.5 kV (positive) or − 3.2 kV (negative).
5
Peptide Quantification by LC-MS/MS
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Peptide quantification by LC-MS/MS was performed on a Thermo Fisher Ultimate 3000 Dionex™ liquid chromatography system and a Thermo Q-Exactive HF-X™ mass spectrometer. The mobile phases consisted of 0.1% formic acid aqueous (mobile phase A) and 0.1% formic acid 80% acetonitrile (mobile phase B) with a gradient of 5–45% over 48 min and a 60 min total gradient. Samples were quantified by A280 absorbance and 1 μg of each was injected. Trypsin samples were run with MS1 settings of 250–1100 m/z window, a resolution of 60,000, AGC target of 5e5, and MIT (maximum inject time) of 54 ms. MS2 scans were collected in data dependent mode with a TopN loop count of 10, resolution of 15,000, AGC target of 1e5, and MIT of 100ms. Chymotrypsin samples were run on the same LC gradient with MS1 settings of 250–1100 m/z window, a resolution of 60,000, AGC target of 1e6, and MIT of 60 ms. MS2 scans were collected in data dependent mode with a TopN loop count of 10, resolution of 15,000, AGC target of 5e5, and MIT of 120ms. Fragmentation was performed with HCD using stepped normalized collision energies (NCE) of 25, 27, 30%94 .
6
Quantitative Proteomics by Nano-LC-MS/MS
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Mass spectrometric analysis was performed using an Ultimate 3000 RSLCnano high-performance liquid peptide separation system (Thermo Scientific, USA) in the nanoflow mode of a Q-Exactive HFX mass spectrometric detector (Thermo Scientific). The chromatographic separation of peptides was carried out on a Peaky C18 reverse phase analytical column (100 μm × 300 mm, 1.7 μm particle size, Molekta, Russia) in a linear elution gradient of mobile phase A (0.1% aqueous formic acid solution) and mobile phase B (80% acetonitrile, 0.1% formic acid) from 2% to 45% at a flow rate of 0.3 μl/min for 60 min, followed by washing the system with 99% B for 5 min and subsequent equilibration of the chromatographic system in the initial gradient conditions (A : B = 2 : 98) for 5 min.
The conditions for mass spectrometric analysis and bioinformatics data processing are detailed in [18] (link).
Each of the proteins presented in the tables was identified in at least three independent experiments.
The conditions for mass spectrometric analysis and bioinformatics data processing are detailed in [18] (link).
Each of the proteins presented in the tables was identified in at least three independent experiments.
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