Q exactive hf x system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Q-Exactive HF-X system is a high-resolution, accurate-mass (HRAM) Orbitrap mass spectrometer designed for advanced qualitative and quantitative analysis. It features high mass resolution, mass accuracy, and sensitivity for complex sample analysis.

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Q Exactive (1165 citations) Q Exactive HF (338 citations) Q Exactive HF-X (212 citations) Q-Exactive system (37 citations) UHPLC-Q Exactive HF-X system (16 citations) Q Exactive HF system (5 citations) Thermo UHPLC-Q Exactive HF-X system (5 citations) Q Exactive HF-X mass spectrometer system (2 citations)

12 protocols using q exactive hf x system

Proteomics and Phosphoproteomics Analysis Pipeline

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The tryptic peptides were dissolved in solvent A (0.1% formic acid and 2% acetonitrile) and then directly extracted with an EASY-nLC 1200 Ultra Performance Liquid Phase System. Solvent B was composed of 0.1% formic acid and 90% acetonitrile. The applied gradient was as follows: 6~22% for more than 38 min, increased to 22~32% over 14 min, increased to 80% over 4 min, and then maintained at 80% for an additional 4 min, all at a fixed flow rate of 450 nL/min.
For ultraperformance liquid chromatography (UPLC), we applied the peptides with a nanospray ionization NSI source and analyzed them on a Q Exactive HF-X system (Thermo) with a 2.0 kV electrospray voltage setting. The peptides were fully scanned at a range of 350 to 1600 m/z and further identified using an Orbitrap at the 120,000-resolution level for proteomics and the 60,000-resolution level for phosphoproteomics. The MS/MS range and resolution were set to 100 m/z and 15,000 (proteomics)/30,000 (phosphoproteomics), respectively. A data-dependent scanning (DDA) program was adopted to acquire the data. The MaxQuant search engine (v.1.5.2.8) was employed to analyze the obtained MS/MS data.

Wang T., Wang H.Q., Yuan B., Zhao G.K., Ma Y.R., Zhao P.S., Xie W.Y., Gao F., Gao W, & Ren W.Z. (2023). Integrative Proteomics and Phosphoproteomics Analysis of the Rat Adenohypophysis after GnRH Treatment. International Journal of Molecular Sciences, 24(4), 3339.

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Peptide Analysis by LC-MS/MS

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Peptides were cleaned up by C18 stage tips, and the concentration was determined (peptide assay, Thermo 23275). The cleaned peptides were dissolved in 0.1% formic acid and analyzed on a Q-Exactive HF-X system coupled with an Easy nanoLC 1200 (Thermo Fisher Scientific, San Jose, CA). Peptides (0.5 μg) were loaded on to an Acclain PepMap RSLC C18 column (250 mm × 75 μm ID, C18, 2 μm, Thermo Fisher). The analytical separation of all peptides was achieved with a 130 min gradient. A linear gradient of 5–30% buffer B over 110 min was executed at a 300 nL/min flow rate followed by a ramp to 100% B in 5 min, and 15 min wash with 100% B, where buffer A was aqueous 0.1% formic acid and buffer B was 80% acetonitrile and 0.1% formic acid. LC–MS experiments were performed in a data-dependent mode with full MS (externally calibrated to a mass accuracy of <5 ppm and a resolution of 60,000 at m/z 200), followed by high-energy collision-activated dissociation-tandem mass spectrometry (MS/MS) of the top 20 most intense ions with a resolution of 15,000 at m/z 200. High-energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 27 eV in the presence of nitrogen bath gas atoms. Dynamic exclusion was 30.0 s. There were two biological replicates for one treatment, and each sample was subjected to two technical LC–MS replicates.

Shen Y., Gao G., Yu X., Kim H., Wang L., Xie L., Schwarz M., Chen X., Guccione E., Liu J., Bedford M.T, & Jin J. (2020). Discovery of First-in-Class Protein Arginine Methyltransferase 5 (PRMT5) Degraders. Journal of medicinal chemistry, 63(17), 9977-9989.

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Terpenoid and Phytohormone Quantification

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The stem and leaf tissues were collected and stored in liquid nitrogen, then transferred to a freezer at −80°C. For the terpenoids detection, stem and leaf samples were preliminarily disposed of by using 2‐chlorophenylalanine (4 ppm) methanol. Next, samples with glass beads were put into the tissue grinder to grind for 90 s at 55 Hz. Following centrifugation at 12000 rpm at 4 °C for 10 min, take the supernatant, filter it through 0.22 μm membrane and transfer the filtrate into the detection bottle before LC‐MS analysis. Then, the sample extracts were analysed using an Ultra Performance Liquid Chromatography (UPLC) Vanquish (Thermo) and Q Exactive HF‐X system (Thermo). For the quantitative detection of phytohormones, stem and leaf tissue samples were used. The self‐construction database which is constructed by reference standards was used to perform qualitative analysis. Additionally, different concentrations of the standards were used to perform quantitative analysis.

Wang S., Liang H., Wang H., Li L., Xu Y., Liu Y., Liu M., Wei J., Ma T., Le C., Yang J., He C., Liu J., Zhao J., Zhao Y., Lisby M., Sahu S.K, & Liu H. (2021). The chromosome‐scale genomes of Dipterocarpus turbinatus and Hopea hainanensis (Dipterocarpaceae) provide insights into fragrant oleoresin biosynthesis and hardwood formation. Plant Biotechnology Journal, 20(3), 538-553.

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Optimized Peptide Fractionation and LC-MS/MS Analysis

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The tryptic peptides were fractionated by Agilent 300 Extend C18 column (Agilent, Santa Clara, CA, USA) using a high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific). For LC-MS/MS analysis, the tryptic peptides were dissolved and analyzed on an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) at a constant flow rate of 450 nl/min. Solvent A (0.1% formic acid) was used to dissolve tryptic peptides, held at solvent B, and increased gradient from 8 to 23% with 0.1% formic acid in 90% acetonitrile.
MS/MS was performed on Q Exactive™ HF-X system (Thermo Fisher Scientific, Waltham, MA, USA). MS1 spectra were collected in the 2.0 kV electrospray voltage and in the range 350–1,600 m/z. For MS/MS, noise-contrastive estimation (NCE) setting as 28, the selected peptides were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z. The HPLC fractionation and LC-MS/MS analysis in our research are supported by Jingjie PTM BioLabs (Hangzhou, China).

Du X., Zhou D., Zhou J., Xue J, & Cheng Z. (2022). Marek's Disease Virus and Reticuloendotheliosis Virus Coinfection Enhances Viral Replication and Alters Cellular Protein Profiles. Frontiers in Veterinary Science, 9, 854007.

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Serum Proteomics Analysis of Heart Diseases

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The proteomics analysis was performed by Novogene Co., Ltd (Beijing, China). The serum samples were labeled with a TMT kit (Thermo Fisher Scientific Inc, MA, USA) and fractionated by high-pH reverse-phase high-performance liquid chromatography with Thermo Q Exactive™ HF-X system. A total of 446 proteins were identified in at least 10 of the 15 serum samples of the ICM, DCM, and control groups (n=5 for each group). The data were standardized using the total sum intensity normalization.13 (link),14 (link)

Huang G., Huang Z., Peng Y., Wang Y., Liu W., Xue Y, & Yang W. (2021). Metabolic Processes are Potential Biological Processes Distinguishing Nonischemic Dilated Cardiomyopathy from Ischemic Cardiomyopathy: A Clue from Serum Proteomics. Pharmacogenomics and Personalized Medicine, 14, 1169-1184.

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UPLC-MS/MS Metabolite Profiling Workflow

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A Vanquish Horizon ultra-high-performance liquid chromatography system (Thermo Scientific) was equipped with ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm; Waters, Milford, USA). The binary gradient elation system consisted of mobile phase (A), which is composed of 95% water and 5% acetonitrile (containing 0.1% Formic acid), and (B), which is composed of 47.5% acetonitrile, 47.5% isopropanol and 5% water (containing 0.1% Formic acid). The separation was achieved using following gradient: 0–100% B over 0–5.5 min, the composition was held at 100% B at 5.5–7.4 min, then 7.4–7.8 min, 100% to 0 B, and 7.8–10 min holding at 0 B. The flow rate was 0.4 mL/min, and the column temperature was 40 °C, the injection volume was 2 µL.
Mass spectrometry was performed on a Q-Exactive HF-X system (Thermo Scientific). The mass range was from m/z 70 to 1050. The resolution was set at 60 000 for the full MS scans and 7500 for MS² scans. The samples were ionized by electrospray and the mass spectrometry operated as follows: spray voltage, 3500 V (positive) and 3500 V (negative); sheath gas flow rate, 50 arbitrary units; auxiliary gas flow rate, 13 arbitrary units; capillary temperature, 325 °C.

Zhang F., Guo R., Cui W., Wang L., Xiao J., Shang J, & Zhao Z. (2021). Untargeted serum metabolomics and tryptophan metabolism profiling in type 2 diabetic patients with diabetic glomerulopathy. Renal Failure, 43(1), 980-992.

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Identification of OsFTIP9 Interactome

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Wild-type and Osftip9 4HA-gOsFTIP9 plants were collected and ground into a fine powder in liquid nitrogen. Total protein was isolated from the samples with RIPA buffer (150-mM NaCl, 50-mM Tris (pH 8.0), 5-mM EDTA (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 1 × protease inhibitor cocktail [Roche, Basel, Switzerland] ). The homogenate was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was collected and incubated with anti-HA agarose beads (Yeasen, Shanghai, China) for 4 h at 4°C. After washing, the immunoprecipitated protein mixture was eluted and analyzed by LC-MS/MS using a Q Exactive HF-X System (Thermo Fisher, Waltham, MA, USA). The spectrum data were searched against the RGAP database using Thermo Proteome Discoverer.

Zhang L., Zhang F., Zhou X., Poh T.X., Xie L., Shen J., Yang L., Song S., Yu H, & Chen Y. (2022). The tetratricopeptide repeat protein OsTPR075 promotes heading by regulating florigen transport in rice. The Plant cell, 34(10).

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Proteomics analysis of virus-infected cells

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Cells infected with MDV and/or REV and the Akt-Flag IP samples were analysed using a high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA) with an Agilent Zorbax 300Extend-C18 column. The tryptic peptides were dissolved on an EASY-nLC 1000 UPLC system (450 nL/min). Tandem mass spectrometry (MS/MS) was performed using a Q Exactive^TM HF-X system (Thermo Fisher Scientific). The MS/MS data were searched in the Uniprot-gallus FASTA database using the Maxquant search engine.
For protein abundance ratios, a 1.2-fold change was taken as the threshold, and a corrected p-value <0.05 was adopted to identify significant changes. To annotate protein pathways, Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/tool/map_pathway2.html) was used.

Du X., Zhou D., Zhou J., Xue J, & Cheng Z. (2022). RIOK3-Mediated Akt phosphorylation facilitates synergistic replication of Marek’s disease and reticuloendotheliosis viruses. Virulence, 13(1), 1184-1198.

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High-Resolution LC-MS/MS Proteomic and Phosphoproteomic Workflow

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LC-MS/MS analyses were performed as reported by Lu et al. (2022) with minor modification. Briefly, after dissolution in mobile phase A (0.1% formic acid, 2% acetonitrile in water, Thermo Fisher Scientific, USA), the peptides were separated using the EASY-nLC 1200 ultra-high-performance liquid system (Thermo Fisher Scientific, USA), followed by ionization in an NSI source and analysis using the Q Exactive^TM HF-X system (Thermo Fisher Scientific, USA). The mass spectrometry scan range was 350–1 600 m/z with a resolution of 120 000 and a second-order mass scan resolution of 30 000. The top 20 peptide parent ions with the highest signal intensity were used for higher-energy collisional dissociation (HCD) fragmentation at a normalized collision energy (NCE) of 28%. Mobile phase B contained 0.1% formic acid and 90% acetonitrile. For proteomic analysis, the elution settings were: 40 min, 6%–22% B; 14 min, 22%–32% B; 3 min, 32%–80% B; 3 min, 80% B, with a constant flow rate of 500 nL/min. For phosphoproteomic analysis, the gradient was as follows: 38 min, 5%–22% B; 15 min 22%–35% B; 4 min 35%–80% B; 3 min, 80% B, with a flow rate of 350 nL/min at 0–53 min and 600 nL/min at 53–60 min.

Niu Y.G., Wei D.B., Zhang X.J., Xu T.S., Li X.Y., Zhang H.Y., An Z.F., Storey K.B, & Chen Q. (2024). Surviving winter on the Qinghai-Xizang Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri. Zoological Research, 45(1), 1-12.

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Nano-LC-MS/MS Analysis of Labeled Peptides

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Labeled peptides were analyzed by online nano flow liquid chromatography tandem mass spectrometry (MS/MS) using the 9RKFSG2_NCS-3500R system (Thermo Fisher Scientific) connected to the Q_Exactive HF-X system (Thermo Fisher Scientific) via a nanoelectrospray ion source. Briefly, a C18-reversed phase column (75 μm × 25 cm, Thermo Fisher Scientific) was equilibrated with solvent A (A: 2% acetonitrile and 0.1% formic acid) and solvent B (B: 80% acetonitrile and 0.1% formic acid). The peptides were eluted using the following gradient: 0–2 min, 0–3% B; 2–92 min, 5–25% B; 92–102 min, 25–45% B; 102–105 min, 45–100% B; 105–120 min, 100–0% B at a flow rate of 300 μL/min. The Q_Exactive HF-X was operated in the data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The survey of full scan MS spectra (m/z 350–1,500) was acquired in the Orbitrap with 70,000 resolutions. The top 20 most intense precursor ions were selected into the collision cell for fragmentation by higher-energy collision dissociation. The MS/MS resolution was set at 35,000 (at m/z 100), with the maximum fill time of 50 ms and a dynamic exclusion of 30 s (Wang et al., 2020a (link),b (link)).

Wang X., Zhang L., Chen H., Wang P., Yin Y., Jin J., Xu J, & Wen J. (2021). Rational Proteomic Analysis of a New Domesticated Klebsiella pneumoniae x546 Producing 1,3-Propanediol. Frontiers in Microbiology, 12, 770109.

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