Q exactive plus hybrid quadrupole orbitrap

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The Q Exactive Plus Hybrid Quadrupole-Orbitrap is a high-resolution mass spectrometer that combines a quadrupole and an Orbitrap analyzer. It is designed to provide accurate and high-resolution mass analysis for a variety of applications.

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13 protocols using q exactive plus hybrid quadrupole orbitrap

Venomous Snake Peptide Identification

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Peptides were separated by nano-liquid chromatography (EASY-nLC 1000, Thermo Fisher Scientific) coupled to a mass spectrometer (Q Exactive Plus Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific) through an EASY-Spray nanoelectrospray ion source (Thermo Fisher Scientific). The MS methods included a full MS scan at a resolution of 70,000 followed by 10 data dependent MS2 scans at a resolution of 17,500. The full MS scan range of 200–2000 m/z was selected, and precursor ions with the charge states of + 1 or greater than + 8 were excluded. Normalized collision energy of HCD fragmentation was set at 28%. Raw LC–MS/MS files were searched using PEAKS Studio version 8.5 against UniProt proteins corresponding to suborder Serpentes and the CDS database predicted by MAKER. A target-decoy approach was used to limit a false discovery rate (FDR) of the identified peptides to less than 1%. Parent and fragment monoisotopic mass errors were set at 10 ppm. Carbamidomethylation of cysteine (C) was used as a fixed modification. Oxidation (M), acetylation (protein N-term), phosphorylation (STY), and deamidation (NQ) were set as variable modifications. A maximum of 1 missed cleavage was allowed.

Saethang T., Somparn P., Payungporn S., Sriswasdi S., Yee K.T., Hodge K., Knepper M.A., Chanhome L., Khow O., Chaiyabutr N., Sitprija V, & Pisitkun T. (2022). Identification of Daboia siamensis venome using integrated multi-omics data. Scientific Reports, 12, 13140.

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Rapid Forensic Drug Identification via Paper Spray MS

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Paper spray mass spectrometry was carried out using a custom‐made paper spray source as per our previous work.²⁵, ³⁰ The paper spray source was coupled to a Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole‐Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Hemel Hempstead, UK). To accommodate fingerprint samples, the analysis substrate (Whatman Grade 1 chromatography paper) was cut into a triangle shape (1.6 × 2.1 cm, base × height).
Positive ion mass spectra were acquired in full scan mode within a range of m/z 50–500 at a mass resolution of 280,000 at m/z 200 (unless otherwise stated), using 4 kV spray voltage, inlet temperature 250°C and S‐lens RF level 50. Standards of cocaine, BZE, heroin and 6‐AM were prepared at 250 ng/mL in ACN, spotted (5 μL) onto the analysis substrate and allowed to dry before being analysed by paper spray for 30 s, 60 s and 120 s, respectively.

Costa C., Frampas C., Longman K.A., Palitsin V., Ismail M., Sears P., Nilforooshan R, & Bailey M.J. (2019). Paper spray screening and liquid chromatography/mass spectrometry confirmation for medication adherence testing: A two‐step process. Rapid Communications in Mass Spectrometry, 35(Suppl 2), e8553.

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Nano-LC-MS/MS Proteomic Analysis of Human Samples

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Peptides were separated by nano-liquid chromatography (EASY-nLC 1000, Thermo Fisher Scientific) coupled to a mass spectrometer (Q Exactive Plus Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific) through an EASY-Spray nanoelectrospray ion source (Thermo Fisher Scientific). The MS methods included a full MS scan at a resolution of 70,000 followed by 10 data-dependent MS2 scans at a resolution of 17,500. The full MS scan range of 200 to 2000 m/z was selected, and precursor ions with the charge states of +1 or greater than +8 were excluded. Normalized collision energy of HCD fragmentation was set at 28%. Raw LC-MS/MS files were searched by X! Tandem (CYCLONE, 2013.2.01) against human databases (ENSEMBL v.76 Homo sapiens GRCh38) plus common contaminants concatenated with their reversed sequences. A target-decoy approach was used to limit a false discovery rate (FDR) of the identified peptides to less than 1%. Parent and fragment monoisotopic mass errors were set at 10 ppm. Carbamidomethyl at cysteine was used as a fixed modification mass. Variable modifications were oxidation at methionine. A maximum of 1 missed cleavage sites was allowed. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride) with the dataset identifier PXD006371.

Pearson L.J., Klaharn I.Y., Thongsawang B., Manuprasert W., Saejew T., Somparn P., Chuengsaman P., Kanjanabuch T, & Pisitkun T. (2017). Multiple extracellular vesicle types in peritoneal dialysis effluent are prominent and contain known biomarkers. PLoS ONE, 12(6), e0178601.

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Label-free LC-MS/MS Analysis of Peptides

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The label-free LC-MS/MS analysis was performed with a Mass Spectrometer (Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™, Thermo Scientific™, Walthem, MA, USA) and an upstream nano-HPLC (Ultimate TM 3000 RSLCnano System, Thermo Scientific, USA). The QExactive Plus was equipped with a Spectroglyph source (Spectroglyph, LLC., Washington, DC, USA), whereby the S-lence was replaced by an ion tunnel [36 (link)]. A 1 µg peptide was injected and separated into fractions using a 60 min acetonitrile gradient of 5–40% and a flow of 300 nL min⁻¹. Two different models of the Acclaim™ PepMap™ 100 C18 (Thermo Scientific, USA) column were used as pre-column and main column. The pre-column had a diameter of 0.3 mm, a length of 5 mm, and a particle size of 5 µm. The diameter of the main column was 0.075 mm by a length of 250 mm and a particle size of 3 µm. The temperature of the column oven was set to 35 °C. Measurements were recorded in data-dependent acquisition mode. On FullMS, the resolution was 70,000, the AGC target was 3 × 10⁶, and the maximum IT was 64 ms. The dd-MS2 settings were 17,500 for the resolution, 2 × 10⁵ for the AGC target, and 100 ms for the maximum IT. TopN was set to 10.

Struck B., Wiersma S.J., Ortseifen V., Pühler A, & Niehaus K. (2024). Comprehensive Proteome Profiling of a Xanthomonas campestris pv. Campestris B100 Culture Grown in Minimal Medium with a Specific Focus on Nutrient Consumption and Xanthan Biosynthesis. Proteomes, 12(2), 12.

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Quantitative LC-MS Analysis of tRNA Modifications

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Total RNA was isolated using Trizol RNA extraction. Small RNAs were subsequently purified from total RNA using the Zymo RNA Clean & Concentrator-5 kit. Small RNAs (1 μg) was digested and analyzed by LC-MS as previously described (Dewe et al., 2017 (link); Zhang et al., 2020 (link); Cai et al., 2015 (link)). Briefly, ribonucleosides were separated using a Hypersil GOLD C18 Selectivity Column (Thermo Scientific) followed by nucleoside analysis using a Q Exactive Plus Hybrid Quadrupole-Orbitrap. The modification difference ratio was calculated using the m/z intensity values of each modified nucleoside following normalization to the sum of intensity values for the canonical ribonucleosides; A, U, G, and C. Statistical analysis of the mass spectrometry results was performed using GraphPad Prism software with error bars representing the standard deviation. Statistical tests and the number of times each experiment was repeated are noted in the figure legend. Raw intensity values for each measured nucleoside are provided in the source data file.

Zhang K., Eldin P., Ciesla J.H., Briant L., Lentini J.M., Ramos J., Cobb J., Munger J, & Fu D. (2024). Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease. eLife, 12, RP90316.

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Affinity Purification and Mass Spectrometric Analysis of NUPR1 Interactome

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The experimental set-up was the same as described previously¹¹. Briefly, MiaPaCa-2 cells, expressing Flag-NUPR1 or GFP-NUPR1 or their controls, were plated in 10 cm² dishes. When MiaPaCa-2 cells expressing Flag-NUPR1 or GFP-NUPR1 reached 70% confluence, they were treated for 24 h and lysed. Equal amounts of total protein were used to incubate with 30 μL of anti-Flag M2-coated beads (MilliporeSigma, F3165) or GFP-Trap Agarose (Chromotek, GTA-10). Beads were then washed 3 times, and proteins were eluted using ammonium hydrogen carbonate buffer containing 0.1 μg/μL of Flag peptide (MilliporeSigma, F3290). Eluted proteins were collected and loaded on NuPAGE 4–12% Bis-Tris acrylamide gels according to the manufacturer’s instructions (Invitrogen). Protein-containing bands were stained with Imperial Blue (Pierce), cut from the gel, and digested with high-sequencing-grade trypsin (Promega) before MS analysis. MS analysis was carried out by LC-MS/MS using an LTQ-Velos-Orbitrap or a Q Exactive Plus Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific) coupled online with a nanoLC Ultimate3000RSLC chromatography system (Dionex). Raw files generated from MS analysis were processed using Proteome Discoverer 1.4.1.14 (Thermo Fisher Scientific).

Santofimia-Castaño P., Huang C., Liu X., Xia Y., Audebert S., Camoin L., Peng L., Lomberk G., Urrutia R., Soubeyran P., Neira J.L, & Iovanna J. (2022). NUPR1 protects against hyperPARylation-dependent cell death. Communications Biology, 5, 732.

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Targeted Proteome Quantification by LC-MS

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Samples were analyzed on an Ultimate 3000 RSLC system coupled to QExactive HF-X hybrid Quadrupole-Orbitrap, QExactive HF hybrid Quadrupole-Orbitrap, or QExactive Plus Hybrid Quadrupole-Orbitrap mass spectrometers (Thermo Fisher Scientific). Acquisition parameters were tuned for the individual experiments and the performance of the mass spectrometer, and can be found in the Source data file. For LFQ, correct peptide ions and peptide ions with amino acid substitutions were targeted in their highest populated charge state. Sets of interference-free fragment chromatograms were extracted using the Skyline software^{72 ,73 (link)} and abundance differences were estimated based on the sum of the individual integrated fragment intensities.

Wohlgemuth I., Garofalo R., Samatova E., Günenç A.N., Lenz C., Urlaub H, & Rodnina M.V. (2021). Translation error clusters induced by aminoglycoside antibiotics. Nature Communications, 12, 1830.

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Serum Metabolomic Profiling by LC-MS

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The LC-MS-based serum metabolic profiling adopted a Vanquish™ Horizon UHPLC system (Thermo Scientific, Germering, Germany) coupled with a Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer. The parameters of chromatography were as follows: column: Ethylene Bridged Hybrid C18 (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, Massachusetts, USA); gradient mobile phase: (A) deionized water containing 0.1% formic acid, (B) acetonitrile/isopropanol (1:1, v/v) mixture containing 0.1% formic acid; flow rate: 0.4 mL/min; sample injection volume: 2 μL; column temperature: 40 °C. The mobile phase gradient was: 0–3 min, A: 95–80%; 3–9 min, A: 80–5%; 9–13 min, A: 5–5%; 13–13.1 min, A: 5–95%; 13.1–16 min, A: 95–95%. The conditions of MS included the scan ranges (M/Z): 70–1050; Sheath gas flow rate (psi): 40; Aus gas flow rate (psi): 10; Aus gas heater temp (°C): 400; Normalized collision energy (V): 20–40–60; and IonSpray Voltage Floating (V): positive mode (ESI⁺), +3500; negative mode (ESI⁻), −2800. A QC sample was inserted for every 6 analytical samples during the experiment to evaluate the stability of the analytical system and assess the reliability of the results.

Huang M., Lin H., Xu C., Yu Q., Wang X., Qin J.G., Chen L., Han F, & Li E. (2020). Growth, Metabolite, Antioxidative Capacity, Transcriptome, and the Metabolome Response to Dietary Choline Chloride in Pacific White Shrimp Litopenaeus vannamei. Animals : an Open Access Journal from MDPI, 10(12), 2246.

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Characterization of HLA-I Peptide Complexes

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C1R were infected at MOI5 for 12hrs and snap frozen as previously^{26 (link)}. HLA class I complexes were isolated as described^{31 (link)}. Briefly, C1R cell pellets were disrupted by cryogenic milling (Retsch Mixer Mill MM 400) and lysed with 0.5% IGEPAL CA-630, 50 mM Tris-HCl pH 8.0, 150 mM NaCl and protease inhibitors (cOmplete Protease Inhibitor Cocktail Tablet; Roche Molecular Biochemicals) for 1 h at 4 °C with rolling. Lysates were cleared by ultracentrifugation and the HLA class I complexes isolated by immunoaffinity purification using pan-HLA class I antibody (W6/32) bound to protein-A-agarose. Peptide-HLA were dissociated with 10% acetic acid and fractionated by reversed-phase high-performance liquid chromatography. Fractions containing peptides were combined into 9–10 pools, vacuum-concentrated, then reconstituted in 15 µl 0.1% formic acid in Optima™ LC-MS water with indexed retention time (iRT) peptides^{77 (link)}. Peptides were analyzed by LC-MS/MS on a Q-Exactive Plus Hybrid Quadrupole Orbitrap (Thermo Fisher Scientific) as described^{24 (link),25 (link)}.

Menon T., Illing P.T., Chaurasia P., McQuilten H.A., Shepherd C., Rowntree L.C., Petersen J., Littler D.R., Khuu G., Huang Z., Allen L.F., Rockman S., Crowe J., Flanagan K.L., Wakim L.M., Nguyen T.H., Mifsud N.A., Rossjohn J., Purcell A.W., van de Sandt C.E, & Kedzierska K. (2024). CD8+ T-cell responses towards conserved influenza B virus epitopes across anatomical sites and age. Nature Communications, 15, 3387.

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Quantitative Analysis of RNA Modifications

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Total RNA was isolated using Trizol RNA extraction. Small RNAs were subsequently purified from total RNA using the Zymo RNA Clean & Concentrator-5 kit. Small RNAs (1 μg) was digested and analyzed by LC-MS as previously described (Dewe et al., 2017 ; Zhang et al., 2020 (link)) (Cai et al, 2015 (link)). Briefly, ribonucleosides were separated using a Hypersil GOLD C18 Selectivity Column (Thermo Scientific) followed by nucleoside analysis using a Q Exactive Plus Hybrid Quadrupole-Orbitrap. The modification difference ratio was calculated using the m/z intensity values of each modified nucleoside following normalization to the sum of intensity values for the canonical ribonucleosides; A, U, G and C. Statistical analysis of the mass spectrometry results was performed using GraphPad Prism software with error bars representing the standard deviation. Statistical tests and the number of times each experiment was repeated are noted in the figure legend. Raw intensity values for each measured nucleoside are provided in the source data file.

Zhang K., Eldin P., Ciesla J.H., Briant L., Lentini J.M., Ramos J., Cobb J., Munger J, & Fu D. (2023). Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease. bioRxiv.

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