Orbitrap q exactive hf x mass spectrometer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, China

The Orbitrap Q Exactive HF-X mass spectrometer is a high-resolution, accurate-mass (HRAM) instrument capable of measuring the mass-to-charge ratio of ions. It combines a quadrupole mass filter with an Orbitrap mass analyzer to provide high mass accuracy and resolving power for a wide range of applications.

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Q Exactive HF mass spectrometer (563 citations) Q Exactive Orbitrap mass spectrometer (430 citations) Q Exactive HF-X mass spectrometer (380 citations) Q Exactive HF Orbitrap mass spectrometer (142 citations) Q Exactive HF Orbitrap (47 citations) Orbitrap Q-Exactive HF-X (13 citations) Q-Exactive HF-X™ mass (6 citations) Orbitrap Q Exactive HF-X spectrometer (3 citations) Orbitrap Q Exactive HF mass (2 citations) Orbitrap Q Exactive HF spectrometer (2 citations)

147 protocols using orbitrap q exactive hf x mass spectrometer

Proteomic Analysis of SLC15A4 by UPLC-MS/MS

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For sample preparation, freshly purified SLC15A4 proteins were subjected to ultra-performance liquid chromatography (UPLC)-MS/MS analysis.
For UPLC-MS/MS, the UPLC system was coupled to a Q-Exactive HFX orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a heated electrospray ionization (HESI) probe. 2 μl protein sample was loaded to a Kinetex® Biphenyl column (2.1 × 150 mm, 2.6 μm, Phenomenex) for the positive mode. Separation was initiated at 80% mobile phase B with a flow rate of 300 μl/min, then the sample was eluted to an orbitrap mass spectrometer with acetonitrile containing 0.1% formic acid as eluent from 80% to 99% within 6.5 min. Untargeted metabolites screening is performed on Q-Exactive HFX orbitrap mass spectrometer (Thermo Fisher Scientific) after calibrating following manufacturer’s guidelines. Data with mass ranges of m/z 300–500 at positive ion mode with data-dependent MS/MS acquisition. The full-scan and fragment spectra were collected with resolutions of 60,000 and 15,000, respectively. The source parameters are as follows: spray voltage: 3,200 V; capillary temperature: 320 °C; heater temperature: 300 °C; sheath gas flow rate: 35 Arb; auxiliary gas flow rate: 10 Arb.
Data analysis was performed by the software Xcalibur 4.4 (Thermo Fisher Scientific) Cholesterol assignment was confirmed using chemical standards based on retention time.

Chen X., Xie M., Zhang S., Monguió-Tortajada M., Yin J., Liu C., Zhang Y., Delacrétaz M., Song M., Wang Y., Dong L., Ding Q., Zhou B., Tian X., Deng H., Xu L., Liu X., Yang Z., Chang Q., Na J., Zeng W., Superti-Furga G., Rebsamen M, & Yang M. (2023). Structural basis for recruitment of TASL by SLC15A4 in human endolysosomal TLR signaling. Nature Communications, 14, 6627.

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Proteomic Analysis by UHPLC-MS/MS

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Use 10 μl of 0.1% FA (formic acid) buffer to resuspend the sample, centrifuge at 10,000 rpm for 5 min at room temperature, and take 1 μg of supernatant sample for proteomics analyses using an EASY-nLCTM 1200 UHPLC (ultra-high pressure liquid chromatography) system (Thermo Fisher, Waltham, Massachusetts, USA) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher, Waltham, Massachusetts, USA) operating in the DDA (data-dependent acquisition) mode. Refer to the previous description for specific details (12 (link)).

Xiao X., Li W., Pan Y., Wang J., Wei Z., Wang S., Wang N., Jian J, & Pang H. (2023). Holistic analysis of lysine acetylation in aquaculture pathogenic bacteria Vibrio alginolyticus under bile salt stress. Frontiers in Veterinary Science, 10, 1099255.

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Serum Metabolite Profiling by LC-MS/MS

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Approximately 200 ul of serum samples were obtained from patients and healthy controls and stored at −80°C for further analysis. The filtrate was injected into the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system after a series of treatments. LC-MS/MS analyses were performed using a Vanquish ultra-high-pressure liquid chromatograph (UHPLC) system (Thermo Fisher, Philadelphia, Massachusetts, USA) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher, Philadelphia, Massachusetts, USA). The raw data files generated by UHPLC-MS/MS were processed using Compound Discoverer 3.0 (CD 3.0, Thermo Fisher, Philadelphia, Massachusetts, USA) to perform peak alignment, peak picking, and quantification for each metabolite. Then, peak intensities were normalized to the total spectral intensity. The normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. The peaks were matched with the mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases to obtain the accurate qualitative and relative quantitative results. Finally, the functional and taxonomic of identified metabolites were annotated through KEGG (http://www.kegg.jp/) (Supplementary File 2).

Wang D., Guo S., He H., Gong L, & Cui H. (2020). Gut Microbiome and Serum Metabolome Analyses Identify Unsaturated Fatty Acids and Butanoate Metabolism Induced by Gut Microbiota in Patients With Chronic Spontaneous Urticaria. Frontiers in Cellular and Infection Microbiology, 10, 24.

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Proteomic Profiling using EASY-nLC™ 1200

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Proteomic analyses were performed using an EASY-nLC™ 1200 UHPLC system (ThermoFisher Scientific, Germany) coupled to an Orbitrap Q Exactive HF-X mass spectrometer (ThermoFisher) operating in the data-dependent acquisition (DDA) mode which was carried out as previously described (Pang et al., 2020 (link)).

Zeng F., Pang H., Chen Y., Zheng H., Li W., Ramanathan S., Hoare R., Monaghan S.J., Lin X, & Jian J. (2021). First Succinylome Profiling of Vibrio alginolyticus Reveals Key Role of Lysine Succinylation in Cellular Metabolism and Virulence. Frontiers in Cellular and Infection Microbiology, 10, 626574.

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Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry

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LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) and an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected onto an Hyperil Gold column (100 mm × 2.1 mm, 1.9 μm) at a flow rate of 0.2 ml/min and separated using a 16 min linear gradient. The eluents for positive polarity mode were eluent A (0.1% formic acid in water) and eluent B (methanol). The eluents for negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows: 2% B 1.5 min, 2%−100% B 12.0 min, 100% B 14.0 min, 100%−2% B 14.1 min, 2% B 16 min. The Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a sheath gas flow rate of 35 arb, an aux gas flow rate of 10 arb, and capillary temperature of 320°C.

Hua X., Zhu J., Yang T., Guo M., Li Q., Chen J, & Li T. (2020). The Gut Microbiota and Associated Metabolites Are Altered in Sleep Disorder of Children With Autism Spectrum Disorders. Frontiers in Psychiatry, 11, 855.

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High-Resolution Mass Spectrometry Proteomics

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Data were acquired using an UHPLC 1290 system coupled to an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Scientific). Peptides were trapped on a column (Maisch Reprosil C18, 3 μm, 2 cm × 100 μm), after which they were separated on an analytical column (Agilent Poroshell EC-C18, 278 μm, 40 cm × 75 μm) using a gradient of 39 min at a column flow of 300 nl min⁻¹. Trapping was performed at 5 μL min⁻¹ for 5 min in solvent A (0.1% formic acid in water) and eluted using as gradient; 13 to 40% solvent B (0.1% formic acid in 80% acetonitrile) in 35 min, 40 to 100% in 3 min and 100% solvent B for 1 min. Full scan MS spectra from m/z 375 to 1600 were acquired at a resolution of 60,000 at m/z 400 after accumulation to an automatic gain control target value of 3e6 ions. Up to 15 most intense precursor ions were selected for fragmentation. HCD fragmentation was performed at normalized collision energy of 27% after the accumulation to a target value of 1e5. MS/MS was acquired at a resolution of 30,000.

Lyu J., Tegelaar M., Post H., Moran Torres J., Torchia C., Altelaar A.F., Bleichrodt R.J., de Cock H., Lugones L.G, & Wösten H.A. (2023). Heterogeneity in Spore Aggregation and Germination Results in Different Sized, Cooperative Microcolonies in an Aspergillus niger Culture. mBio, 14(1), e00870-22.

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UHPLC-Orbitrap MS Analysis of Compounds

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LC−MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected onto a Hypersil Gold column (100 mm × 2.1 mm, 1.9 μm) using a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; and 2% B, 16 min. The Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 arb and an aux gas flow rate of 10 arb.

Yang H., Han T., Wu Y., Lyu L., Wu W, & Li W. (2023). Quality analysis and metabolomic profiling of the effects of exogenous abscisic acid on rabbiteye blueberry. Frontiers in Plant Science, 14, 1224245.

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DIA-based Quantitative Proteomics Workflow

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Each serum sample peptide was reconstituted in 0.1% FA, mixed with 0.2 µL standard peptides (iRT kit, Biognosys), and injected into the EASY-nLCTM 1200 UHPLC system coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) operating in DIA mode. The liquid conditions were the same as those of the DDA model for library construction. For DIA acquisition, the MS1 resolution was set to 60,000 (at 200 m/z), and the MS2 resolution was set to 30,000 (at 200 m/z). The m/z range covered from 350 to 1,500 m/z and was separated into 30 acquisition windows (Table S1). The full scan AGC target was set to 3×10⁶, with an injection time of 50 ms. DIA settings included NCE of 27%, a target value of 1×10⁶, and an automatic maximum injection time was set to automatic to allow the MS to continuously operate in the parallel ion filling and detection mode.

Jia B., Zhao X., Wu D., Dong Z., Chi Y., Zhao J., Wu M., An T., Wang Y., Zhuo M., Li J., Chen X., Tian G., Long J., Yang X., Chen H., Wang J., Zhai X., Li S., Li J., Ma M., He Y., Kong L., Brcic L., Fang J, & Wang Z. (2021). 放Identification of serum biomarkers to predict pemetrexed/platinum chemotherapy efficacy for advanced lung adenocarcinoma patients by data-independent acquisition (DIA) mass spectrometry analysis with parallel reaction monitoring (PRM) verification. Translational Lung Cancer Research, 10(2), 981-994.

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Shotgun Proteomics with Orbitrap Q Exactive HF-X

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An EASY-nLC™ 1200 UHPLC system (Thermo Fisher Scientific, Germany) coupled to an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Germany) was used to perform Shotgun proteomics analyses in the data-dependent acquisition (DDA) mode. The specific details have been described previously (Pang et al., 2019 (link)).

Wang J., Pang H., Yin L., Zeng F., Wang N., Hoare R., Monaghan S.J., Li W, & Jian J. (2022). A Comprehensive Analysis of the Lysine Acetylome in the Aquatic Animals Pathogenic Bacterium Vibrio mimicus. Frontiers in Microbiology, 13, 816968.

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High-Resolution LC-MS/MS for Metabolite Analysis

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LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected onto an Hyperil Gold column (100 × 2.1 mm, 1.9 μm) using a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (Methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2-100 % B, 12.0 min; 100 % B, 14.0 min; 100-2 % B, 14.1 min; 2 % B, 16 min. Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320 °C, sheath gas flow rate of 35 arb and aux gas flow rate of 10 arb.

Xiao H.W., Cui M., Li Y., Dong J.L., Zhang S.Q., Zhu C.C., Jiang M., Zhu T., Wang B., Wang H.C, & Fan S.J. (2020). Gut microbiota-derived indole 3-propionic acid protects against radiation toxicity via retaining acyl-CoA-binding protein. Microbiome, 8, 69.

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