LC-MS/MS analyses were performed on a Q-Exactive HF high-resolution mass spectrometer (Thermo) with a nano-Acquity UPLC system (Waters) and a nano-electrospray ionization source fitted with a SilicaTip emitter (New Objective). Samples were trapped on a 2D Symmetry C18 trapping column with dimensions 180 μm x 20 mm and particle diameter of 5 μm, pore size 100 Å. The trapping time was 5 minutes at 5 μL/minute (99.9:0.1 v/v water/ACN 0.1% formic acid). The samples were separated on a 75 μm x 250 mm high strength silica (HSS) T3 column with 1.8 μm particle diameter (Waters) heated to 55 °C. Peptides were eluted using a gradient of 3–30% acetonitrile with 0.1% formic acid over 90 minutes at a flow rate of 0.3 μL/min. LC-MS/MS data were collected using a top 20 data-dependent acquisition (DDA) method including MS1 at 120k and MS2 at 30k resolution. The AGC target for MS1 was 3 × 106 ions with a max IT of 50 msec. The AGC target for MS2 was 1 × 105 ions with a max IT of 45 msec. The normalized collision energy (NCE) was set to 30 V and the scan range was 375–1600 m/z. The isolation window was 0.7 m/z and the dynamic exclusion time was set to 20.0 seconds.
Q exactive hf high resolution mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q Exactive HF is a high-resolution mass spectrometer designed for advanced analytical applications. It features high-performance quadrupole technology and a high-field Orbitrap mass analyzer, enabling accurate mass measurements and detailed structural information. The core function of the Q Exactive HF is to provide sensitive and precise mass analysis of a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
Waters 2d uplc, by Waters Corporation (3 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
Silicatip emitter, by New Objective (1 mentions)
Hss t3 column, by Waters Corporation (1 mentions)
Q exactive high resolution mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Triversa nanomate, by Thermo Fisher Scientific (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
6 protocols using q exactive hf high resolution mass spectrometer
1
High-Resolution LC-MS/MS Proteomics Protocol
2
Direct Infusion Mass Spectrometry for Blood Metabolomics
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Data acquisition was performed using our in-house pipeline for direct infusion mass spectrometry, as described by (de Sain-van der Velden et al., 2017 (link)). In short, from each blood spot card a 3 mm disk was punched and extracted in an ultrasonic bath with acetonitrile, formic acid and internal standards. Run order was randomised based on farm and, if multiple countries were measured simultaneously, country.
Following extraction, the samples were filtered and subjected to chip-based nano electrospray DI-MS analysis in positive and negative mode using an Advion TriVersa Nanomate combined with a Thermo Scientific Q-Exactive HF high resolution mass spectrometer. In positive and negative ion modes, the signal was collected for 3 min and 1.5 min, respectively, using a Thermo Scientific Q Exactive high resolution mass spectrometer. Three technical replicates were measured per blood spot and the resulting signal was saved as a.raw Thermo file for each replicate. The raw files and metadata including country, breed, temperature at sampling, sex, and feed type is hosted on MetaboLights alongside the raw data.
Following extraction, the samples were filtered and subjected to chip-based nano electrospray DI-MS analysis in positive and negative mode using an Advion TriVersa Nanomate combined with a Thermo Scientific Q-Exactive HF high resolution mass spectrometer. In positive and negative ion modes, the signal was collected for 3 min and 1.5 min, respectively, using a Thermo Scientific Q Exactive high resolution mass spectrometer. Three technical replicates were measured per blood spot and the resulting signal was saved as a.raw Thermo file for each replicate. The raw files and metadata including country, breed, temperature at sampling, sex, and feed type is hosted on MetaboLights alongside the raw data.
3
High-Resolution LC-MS Metabolite Profiling
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Waters 2D UPLC (Waters, Milford, CT, United States) tandem Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to separate and detect metabolites (see Supplementary Material for mobile phase and chromatographic separation). Samples (5 μl) were detected on a BEH C18 column (1.7 μm 2.1*100 mm, Waters, United States) at 45°C at a flow rate of 0.35 ml/min. Q Exactive HF mass spectrometer (Thermo Fisher Scientific, United States) was used for primary and secondary mass spectrometry data acquisition. Primary and secondary resolutions are 120,000 and 30,000, respectively. The samples were analyzed in positive ion (Spray voltage was 3.8 kV) and negative ion (Spray voltage was 3.2 kV) modes. The mass scanning range was 70–1050 m/z, and the ion transfer tube temperature was 320°C. The flow rates of sheath gas and auxiliary gas were set to 40 L/min and 10 L/min, respectively.
4
Untargeted Metabolite Profiling by UPLC-QTOF
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Waters 2D UPLC (Waters, Milford, CT, United States) tandem Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to separate and detect metabolites. Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters, USA) was used for chromatographic separation. Q Exactive HF mass spectrometer (Thermo Fisher Scientific, United States) was used for primary and secondary mass spectrometry data acquisition. The samples were analyzed in positive ion (spray voltage was 3.8 kV) and negative ion (spray voltage was 3.2 kV) modes. The mass scanning range was 70–1,050 m/z. The flow rates of sheath gas and auxiliary gas were set to 40 L/min and 10 L/min, respectively.
5
Comprehensive Lipidomics Sequencing Analysis
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Lipidomics sequencing analysis was performed by BGI Genomics Co. Ltd. (Shenzhen, China). Firstly, lipid metabolites were extracted, and quality control (QC) samples were prepared. Waters 2777C UPLC (Waters, USA) and Q Exactive HF High-resolution mass spectrometer (Thermo Fisher Scientific, USA) were used for the separation and detection of lipid metabolites. Data were then imported into LipidSearch v.4.1 software (Thermo Fisher Scientific, USA) for mass spectrometry analysis, and the lipid molecular identification results and quantitative results were obtained. Finally, the above data were used for data quality control, global metabolite analysis, screening of differentially expressed lipids between groups and unsaturation analysis of lipid molecules.
6
Untargeted Metabolomics Analysis of Brain Tissues
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The untargeted metabolomics analysis process was performed using the BGI-tech pipeline (BGI). Briefly, 25 mg of brain tissues was weighed and extracted by directly adding 800 µl of precooled extraction reagent, and internal standards mix 1 (IS1) and internal standards mix 2 (IS2) were added for quality control of sample preparation. After homogenization for 5 min using TissueLyser (JXFSTPRP, China), the samples were sonicated for 10 min and incubated for 1 hour at -20°C. Then, the samples were centrifuged for 15 min (25000 rpm, 4 °C), and the supernatant was transferred for vacuum freeze-drying. The metabolites were resuspended in 200 µl of 10% methanol and sonicated for 10 min at 4 °C, after centrifugation for 15 min at 25000 rpm, and the supernatants were transferred to autosampler vials for LC-MS analysis. A quality control (QC) sample was prepared by pooling the same volume of each sample to evaluate the reproducibility of the whole LC-MS analysis. This experiment used a Waters 2 D UPLC (Waters, USA) tandem Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific, USA) for the separation and detection of metabolites.
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