Q exactive plus hybrid quadrupole orbitrap mass spectrometer

Manufactured by Thermo Fisher Scientific

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The Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer is a high-performance analytical instrument designed for advanced mass spectrometry applications. It combines a quadrupole mass analyzer with an Orbitrap mass analyzer, providing high mass resolution, accurate mass measurements, and sensitive detection of a wide range of analytes.

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161 protocols using q exactive plus hybrid quadrupole orbitrap mass spectrometer

Protein Extraction and Trypsin Digestion

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The methods of protein extraction and trypsin digestion were performed according to the protocol reported in Sun et al. (2017) (link) with slight modifications. The gradient was comprised of an increase from 6 to 22% of solvent B (0.1% FA in 98% ACN) for 40 min, from 22 to 35% over 12 min, climbing to 80% over 4 min, and then holding at 80% for the last 4 min. A constant flow rate of 300 nL/min was maintained on an EASY-nLC 1000 UPLC system, and the resulting peptides were analyzed by a Q Exactive^TM plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).
The peptides were subjected to Nanospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM plus (Thermo) coupled with an online Ultra Performance Liquid Chromatography (UPLC). For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.

Yang Q., Li Y., Apaliya M.T., Zheng X., Serwah B.N., Zhang X, & Zhang H. (2018). The Response of Rhodotorula mucilaginosa to Patulin Based on Lysine Crotonylation. Frontiers in Microbiology, 9, 2025.

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Peptide Separation and Analysis by LC-MS

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Three parallel analyses for each fraction were performed. Peptides were dissolved in 0.1% formic acid (FA), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% FA in 98% acetonitrile) for 24 min, 23% to 35% for 8 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 280 nl/min on an EASY-nLC 1000 UPLC system, the resulting peptides were analyzed by Q Exactive^TM Plus hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).

Sun X., Li Z., Liu H., Yang J., Liang W., Peng Y.L, & Huang J. (2017). Large-scale identification of lysine acetylated proteins in vegetative hyphae of the rice blast fungus. Scientific Reports, 7, 15316.

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Reversed-Phase Liquid Chromatography and High-Resolution Mass Spectrometry for Peptide Analysis

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Peptides were dissolved in solvent A (0.1% FA in 2% ACN) and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) with a linear gradient of 8–35% solvent B (0.1% FA in 98% ACN) for 12 min and 35–80% solvent B for 4 min at a constant flow rate of 320 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analysed by Q Exactive^TM Plus hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher Scientific).
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM Plus (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 30% NCE; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 10 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5000 in the MS survey scan with 5.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5 E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800.

Pei H., He L., Shao M., Yang Z., Ran Y., Li D., Zhou Y., Tang M., Wang T., Gong Y., Chen X., Yang S., Xiang M, & Chen L. (2018). Discovery of a highly selective JAK3 inhibitor for the treatment of rheumatoid arthritis. Scientific Reports, 8, 5273.

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Reversed-Phase Liquid Chromatography-MS

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The enriched peptides were dissolved in 0.1% formic acid (v/v) and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was comprised of an increase from 6% to 23% solvent B [0.1% formic acid (v/v) in 98% acetonitrile (v/v)] for 24 min, 23% to 35% for 8 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 280 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by Q Exactive^TM Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).

Reverdy A., Chen Y., Hunter E., Gozzi K, & Chai Y. (2018). Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity. PLoS ONE, 13(9), e0204687.

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Reversed-Phase Peptide Separation and Mass Spectrometry Analysis

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Peptides were dissolved in solvent A (0.1% FA in 2% ACN), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMapRSLC, Thermo Scientific) with a linear gradient of 5–25% solvent B (0.1% FA in 98% ACN) for 50 min, 25–35% solvent B for 10 min, and 35–80% solvent B for 10 min at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by Q Exactive TMPlus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 28% NCE; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 10 MS/MS scans was applied for the top 10 precursor ions above a threshold ion count of 2E4 in the MS survey scan with 5.0s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1600 Da.

Xu H., Chen X., Ying N., Wang M., Xu X., Shi R, & Hua Y. (2017). Mass spectrometry-based quantification of the cellular response to ultraviolet radiation in HeLa cells. PLoS ONE, 12(11), e0186806.

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Phosphorylation Site Identification of RRM1

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RRM1 S631 phosphorylation was identified using liquid chromatography/tandem mass spectrometry (LC-MS/MS) by PTM-Biolabs Inc. (Hangzhou, China). First, Flag-RRM1 was overexpressed in H1299 cells and immunoprecipitated by Flag, and was subjected to SDS-PAGE, followed by cutting the Flag-RRM1 band. Then, the in-gel tryptic digestion was performed and peptides were extracted, dried, and resuspended in 2% acetonitrile /0.1% formic acid. The peptides were then analyzed by Q-ExactiveTM plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). The resulting MS/MS data were processed using mascot search engine (v.2.3.0) and tandem mass spectra were searched against RRM1.

Tian L., Chen C., Guo Y., Zhang F., Mi J., Feng Q., Lin S., Xi N., Tian J., Yu L., Chen Y., Cao M., Lai C., Fan J., Zhang Y, & Chen G. (2021). mTORC2 regulates ribonucleotide reductase to promote DNA replication and gemcitabine resistance in non-small cell lung cancer. Neoplasia (New York, N.Y.), 23(7), 643-652.

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Proteomic Analysis of ESCC Tumor Samples

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Samples of three ESCC tumor and corresponding normal tissues have been collected. Samples were first sonicated on ice using a high intensity ultrasonic processor in lysis buffer. The protein concentration was determined by 2-D Quant kit according to the manufacturer’s instructions. Amount of protein(100 μg) from each sample was digested with trypsin for the following experiments. The protein samples were labeled with the TMT tags and then fractionated by high pH reverse-phase HPLC using Agilent 300Extend C18 column. Peptides were dissolved with 0.1% formic acid and then directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). The separation of peptide was performed using a reversed-phase analytical column. The resulting peptides were then analyzed by Q ExactiveTM plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM plus (Thermo) coupled online to the UPLC. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.

Cai R., Wang P., Zhao X., Lu X., Deng R., Wang X., Su Z., Hong C, & Lin J. (2020). LTBP1 promotes esophageal squamous cell carcinoma progression through epithelial-mesenchymal transition and cancer-associated fibroblasts transformation. Journal of Translational Medicine, 18, 139.

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Reversed-phase LC-MS Peptide Analysis

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Peptides were dissolved in 0.1% FA and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). To separate peptide fractions, a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) was used, with gradient of an increase in solvent B (0.1% FA in 98% ACN) from 6% to 22% for 26 min, 22% to 35% for 8 min, went up to 80% in 3 min and staying at 80% for another 3 min. The whole process happened at a constant flow rate of 300 nL/min on an EASY-nLC 1000 UPLC system. The following analysis of resulting peptides was carried out by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).

Jiang M., Hua Z., Dong Y., Liu Z., Thiele C.J, & Li Z. (2018). Quantitative ubiquitylome analysis and crosstalk with proteome/acetylome analysis identified novel pathways and targets of perifosine treatment in neuroblastoma. Translational cancer research, 7(6), 1548-1560.

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Optimized Peptide Separation and MS Analysis

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The peptides were dissolved in 0.1% solvent A (formic acid, FA), directly loaded onto a reversed-phase pre-column (Thermo Scientific, Acclaim PepMap 100, USA). Peptide separation was performed using a reversed-phase analytical column (Thermo Scientific, Acclaim PepMap RSLC). The gradient was increased with solvent B (0.1% FA, 90% ACN) from 8% to 26% in 22 min, from 26% to 40% in 12 min, and increased to 80% in 3 min to remain at 80% for the last 3 min. This was carried out at a constant flow rate of 400 nL/min using an EASY-nLC 1000 UPLC system. The peptides were then analyzed on a Q Exactive^TM plus hybrid quadrupole-orbitrap mass spectrometer (ThermoFisher Scientific, USA).
The peptides were analyzed via the Q ExactiveTM plus (Thermo Scientific) with a positive ion model and data-dependent acquisition. The resolution of the MS scan was 70,000, and the ion fragment was 17,500. Based on the MS scan, the top 20 precursor ions were selected to fragment with 30 s of dynamic exclusion. The electrospray voltage applied was 2.0 kV. The MS/MS spectra were generated using automatic gain control (AGC) to prevent overfilling of the Orbitrap and accumulation of 5E4 ions. For the MS scans, the m/z scan range was set from 350 to 1800. The first fixed mass was set at 100 m/z.

Ren C., Chen Y., Tang J., Wang P., Zhang Y., Li C., Zhang Z, & Cheng X. (2023). TMT-Based Comparative Proteomic Analysis of the Spermatozoa of Buck (Capra hircus) and Ram (Ovis aries). Genes, 14(5), 973.

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Quantitative Mass Spectrometry Analysis

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Peptides were dissolved in 0.1% FA, and directly loaded onto a reversed-phase precolumn (Acclaim PepMap RSLC columns, Thermo Fisher Scientific, 81 Wyman Street, Waltham, MA, USA). The resulting peptides were analyzed via Q ExactiveTM plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM plus (Thermo) coupled online to the UPLC. Finally, MS/MS data were obtained using Mascot (v.2.3.0) [45 (link)], and the sequenced proteins were quantified. The quantitative ratio ≥ 1.2 was regarded as up-regulated, while the ratio ≤ 0.83 was regarded as down-regulated.

Chen D., Guo X., Wang K., Zhao W., Chang Z., Wang Q, & Xu C. (2023). Multi-Omics Analysis Reveals the Pathogenesis of Growth-Disordered Raccoon Dog. International Journal of Molecular Sciences, 24(18), 14237.

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