Q exactive mass spectrometer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, China, United Kingdom, Japan, Denmark, Uruguay

The Q Exactive mass spectrometer is a high-resolution, accurate-mass (HRAM) instrument designed for advanced proteomics, metabolomics, and small molecule applications. It combines a quadrupole mass filter with a high-field Orbitrap mass analyzer to provide precise mass measurements and high-quality data.

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1 635 protocols using q exactive mass spectrometer

Quantitative Proteome Analysis by LC-MS/MS

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The LC/MS-MS analysis was conducted using an Easy nLC system (Thermo Fisher Scientific, Waltham, USA) coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific). For the analysis, 6 μL digested peptides were utilized. The mobile phases consisted of 0.1% formic acid aqueous solution for phase A and a mixture of 0.1% formic acid and 84% acetonitrile (ACN) (Merck, Darmstadt, Germany) aqueous solution for phase B. Peptides were loaded onto a C18 reversed-phase column (Thermo Scientific EASY column, 100 μm × 2 cm, 5 μm-C18) and separated in an analytical column (Thermo scientific EASY column, 75 μm × 10 cm, 3 μm-C18) at a flow rate of 300 nL/min. The liquid-phase gradient was set as follows: from 0% B to 55% B over 0–110 min, from 55% B to 100% B over 110–115 min, and 100% B at 115–120 min.
Peptide separations were analyzed using a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) by dynamically choosing the 20 most abundant ions from one full mass scan (300–1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. The normalized collision energy was set at 27 eV, and dynamic exclusion was applied for 60 s. The underfill ratio was defined as 0.1%. The resolution of the first level of the mass spectrum was set at 70,000 at m/z 200, while the second level was set at 17,500 at m/z 200. The automatic-gain-control (AGC) target was set to 3e6.

Pan C., He X., Xia L., Wei K., Niu Y, & Han B. (2024). Proteomic Analysis of Salivary Secretions from the Tea Green Leafhopper, Empoasca flavescens Fabrecius. Insects, 15(4), 296.

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Liquid Chromatography-Mass Spectrometry Proteomics

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An Eksigent NanoLC system (nano2D ultra) coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.) was used for analysis. Tryptic peptides were reconstituted in 0.1% (vol/vol) formic acid to approximately 0.25 μg/μL, and 1 μg of peptides was loaded. The column used for peptide separation was of 75-μm inner diameter and was 15 cm long, packed with reverse-phase C₁₈ resin (1.9-μm/120-Å ReproSil-Pur C₁₈ resin; Dr. Maisch GmbH). A 90-min gradient with acetonitrile changing from 5% to 30% (vol/vol) was used, at a flow rate of 300 nL/min. Solvent A was composed of 0.1% (vol/vol) formic acid, and solvent B was composed of 0.1% (vol/vol) formic acid and 80% (vol/vol) acetonitrile. MS analysis was performed with a Q Exactive mass spectrometer (Thermo Fisher Scientific Inc.). Full MS scans were performed from m/z 300 to m/z 1,800, and data-dependent MS/MS scans were performed for the 12 most intense ions. MS and MS/MS scans were performed with resolutions of 70,000 and 17,500, respectively. Samples were loaded in a randomized order. In this study, 197 samples were analyzed over a period of 23 days. All raw data from LC-MS/MS have been deposited with the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE (41 (link)) partner repository via the data set identifier PXD030458.

Sun Z., Wang W., Li L., Zhang X., Ning Z., Mayne J., Walker K., Stintzi A, & Figeys D. (2022). Comprehensive Assessment of Functional Effects of Commonly Used Sugar Substitute Sweeteners on Ex Vivo Human Gut Microbiome. Microbiology Spectrum, 10(4), e00412-22.

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Metabolomic and Lipidomic Profiling using UHPLC-MS

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A Vanquish UHPLC system (Thermo Fisher) was coupled to a Q Exactive mass spectrometer (Thermo Fisher) for oxylipins analysis, and an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher) for metabolomics analysis. Metabolites were resolved across a 2.1 × 150 mm, 1.7-µm Kinetex SB-C18 column (Phenomenex) using a 5-min, reverse-phase gradient from a previously described method^{57 (link)}. For oxylipins, the samples were analysed using a 7-min gradient across a 1.7-µm, 2.1 × 100 mm Acquity UPLC BEH column (Waters). The run order of samples was randomized and technical replicates were included to assess quality control. Raw files were converted to .mzXML files using RawConverter. The resultant files were processed with El-Maven (Elucidata) alongside the KEGG database for metabolite assignment and peak integration as previously described^{58 (link)}. Lipidomics analysis used a Vanquish UHPLC system (Thermo Fisher) coupled to a Q Exactive mass spectrometer (Thermo Fisher). The samples were randomized and resolved across a 2.1 × 30 mm, 1.7-µm Kinetex C18 column (Phenomenex) using a 5-min reverse-phase gradient adapted from a previous method^{59 (link)}. Technical replicates were included to assess quality control. Lipid assignments and peak integration were performed using LipidSearch v 5.0 (Thermo Fisher).

Redondo-Muñoz M., Rodriguez-Baena F.J., Aldaz P., Caballé-Mestres A., Moncho-Amor V., Otaegi-Ugartemendia M., Carrasco-Garcia E., Olias-Arjona A., Lasheras-Otero I., Santamaria E., Bocanegra A., Chocarro L., Grier A., Dzieciatkowska M M., Bigas C., Martin J., Urdiroz-Urricelqui U., Marzo F., Santamaria E., Kochan G., Escors D., Larrayoz I.M., Heyn H., D’Alessandro A., Attolini C.S., Matheu A., Wellbrock C., Benitah S.A., Sanchez-Laorden B, & Arozarena I. (2023). Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy. Nature Metabolism, 5(9), 1544-1562.

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Peptide Identification via LC-MS/MS

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The digested peptide mixtures were subjected to FASP enzymatic digestion. Following desalting, liquid chromatography–tandem mass spectrometry (LC–MS/MS) was performed using a Q‐Exactive mass spectrometer coupled with an Easy nLC (Thermo Fisher Scientific). The peptide sample was first loaded onto a C18‐reversed‐phase analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 μm × 15 cm, nano viper, P/N164943) in buffer A (0.1% formic acid in high‐performance liquid chromatography grade water) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min. A linear chromatographic gradient was achieved with a linear increase in buffer B percentage, which was set up as follows: 6% buffer B for 5 min, 6%–28% buffer B for 40 min, 28%–38% buffer B for 5 min, 38%–100% buffer B for 5 min, and hold in 100% buffer B for 5 min. The peptide was then added to a Q Exactive mass spectrometer (Thermo Fisher Scientific). MS analysis was performed for 60 min in positive ion mode.

Huang Q., Li X., Sun J, & Zhou Y. (2022). Tumor‐derived endomucin promotes colorectal cancer proliferation and metastasis. Cancer Medicine, 12(3), 3222-3236.

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Camellia Husks Extract Profiling

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Camellia husks extract was recorded and analysed by Liquid Chromatography-Mass Spectrometry. Using an Ultimate 3000 LC (column. Hypersil GOLD 100 × 2.1 mm, 3 μm Thermo Scientific, T = 251, wavelength: 190–400 nm, U = 0.25 ml min⁻¹, mobile phase: A: methanol B: 0.1% formic acid solution) and a Thermo Scientific Q Exactive mass spectrometer (spray voltage: 3200 V) with gradient elution. T = 0-10-15-15.1-22, A% = 15-90-90-15-15) and Thermo Scientific Q Exactive mass spectrometer (spray voltage: 3200 V, capillary temperature. 300.00, intrathecal gas: 40.00 Arb, auxiliary gas: 8.00 Arb, maximum spray current.

Chen J., Ni Y., Mei B., Jiang H., Wang Y., Chen Y, & Qi H. (2022). Extraction of pigments from camellia seed husks and their application on silk fabrics. RSC Advances, 12(53), 34715-34723.

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LC-MS/MS Peptide Analysis Protocol

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Peptide samples (900 ng for each) were reconstituted in 0.1% FA/H₂O and analyzed by LC‐MS/MS with an Easy n‐LC 1000 HPLC and a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA). The peptides were separated with a C18 column (75 µm × 20 cm) packed with Reprosil‐Pur C18 AQ particles (3.0 μm, Dr. Maisch HPLC GmbH) with solvent A (0.1% FA) and solvent B (ACN/0.1% FA) at a flow rate of 280 nl/min with a gradient: 4% B (0 min), 8% B (5 min), 22% (58 min), 32% B (70 min), 90% B (71 min), and 90% B (78 min).
A Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) was used for MS analysis in data‐dependent acquisition mode. Each MS1 spectrum was obtained at 70,000 high‐resolution (m/z 200) at 300–1600 m/z. The automatic gain control (AGC) target value was 3E6 for a maximum filling time of 60 ms. The top 20 most abundant precursor ions were selected with a 2.0‐m/z isolation window and fragmented with a normalized collision energy of 27. MS/MS spectra were acquired at 17,500 resolution (m/z 200) with a 50,000 target value over a maximum injection time of 80 ms by setting up an isolation window of 2.0 m/z and dynamic exclusion time of 40 s.

Zhang T., Chen X., Sun L., Guo X., Cai T., Wang J., Zeng Y., Ma J., Ding X., Xie Z., Niu L., Zhang M., Tao N, & Yang F. (2021). Proteomics reveals the function reverse of MPSSS‐treated prostate cancer‐associated fibroblasts to suppress PC‐3 cell viability via the FoxO pathway. Cancer Medicine, 10(7), 2509-2522.

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Liquid Chromatography-Mass Spectrometry Proteomics

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Proteomic Workflow for Peptide Identification

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The protein solution was firstly digested into a peptide mixture with protease (Ragelle et al., 2017 (link)). Then, LC-MS/MS was performed using a Q Exactive mass spectrometer coupled with an Easy nLC (Thermo Fisher Scientific, United States). The peptide sample was loaded onto the C18-reversed phase analytical column (Thermo Fisher Scientific, United States) in buffer A (0.1% formic acid in HPLC grade water), and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) with a flow rate at 300 nL/min. The linear chromatographic gradient was achieved with linear increase of buffer B percentage. After that, the peptide entered into the Q Exactive mass spectrometer (Thermo Fisher Scientific, United States). The MS analysis was set for 60 min in a positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the full scan (350–1,800 m/z) for HCD fragmentation. The raw data obtained was then imported into Proteome Discoverer 2.2 (Thermo Fisher Scientific, United States) for protein identification, then embedded Mascot 2.6 engines was used for database searches. Protein identification was performed using reviewed database.

Zhang J., Xiang Y., Yang Q., Chen J., Liu L., Jin J, & Zhu S. (2024). Adipose-derived stem cells derived decellularized extracellular matrix enabled skin regeneration and remodeling. Frontiers in Bioengineering and Biotechnology, 12, 1347995.

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Spectroscopic Characterization of Natural Products

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An electrospray ionization source (ESI)-equipped Q-Exactive Mass spectrometer (Thermo Fisher Scientific Corporation, Waltham, MA, USA) was used to analyse the HR-ESI-MS data. A Shimadzu UV-260 spectrometer (Shimadzu Corporation, Tokyo, Japan) and a Perkin–Elmer 683 infrared spectrometer (PerkinElmer, Inc., Waltham, MA, USA) were used to obtain the UV and IR spectra, respectively. A JASCO P-200 polarimeter (JASCO Corporation, Tokyo, Japan) with a 5 cm cell was applied to measure the optical rotation value. The NMR spectra with TMS as the internal standard were taken on a Brucker Avance III 600 FT NMR spectrometer (Bruker Corporation, Billerica, MA, USA).
Column chromatography was performed with silica gel (Yantai Chemical Industry Research Institute, Shandong, China), Cosmosil 75 C18-MS-II (75 μm, Nacalai Tesqye corporation, Kyoto, Japan), and Spehadex LH-20 (GE Healthcare, Danderyd, Sweden). Semi-preparative HPLC was conducted on an Aglient HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) system equipped with a diode array detector via a preparative Cosmosil ODS column. The HR-ESI-MS spectra were measured using a thermo Q-Exactive Mass spectrometer (Thermo Fisher Scientific Corporation).

Liu S.Z., Xu G.X., He F.M., Zhang W.B., Wu Z., Li M.Y., Tang X.X, & Qiu Y.K. (2022). New Sorbicillinoids with Tea Pathogenic Fungus Inhibitory Effect from Marine-Derived Fungus Hypocrea jecorina H8. Marine Drugs, 20(3), 213.

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Negative Mode Nano-ESI LC-MS Analysis

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Reaction solutions were vacuum-filtered using 0.22-μm, PVDF filter plates (Millipore, USA), and then prepared in 50% methanol before being injected to a nano-ESI source on a Q-Exactive mass spectrometer (Thermo Scientific, USA) with a disposable pico-emitter. Samples were analyzed in a negative mode at a spray voltage of 2.5 kV, capillary temperature of 250 °C, automatic gain control target of 1 × 10⁶, injection time of 100 ms, and resolution of 140,000. Spectra were analyzed using Qual Browser in Thermo Xcalibur (v2.2) software (Thermo Scientific, USA). For LC–MS analysis, the filtered samples were analyzed using a Q-Exactive mass spectrometer (Thermo Scientific, USA), equipped with an Ultimate 3000 HPLC system (Thermo Scientific, USA) and a Hypersil GOLD column (50 × 2.1 mm) (Thermo Scientific, USA).

Vuong T.V, & Master E.R. (2020). Enzymatic production of 4-O-methyl d-glucaric acid from hardwood xylan. Biotechnology for Biofuels, 13, 51.

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