Q exactive hf orbitrap

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The Q Exactive HF Orbitrap is a high-resolution, high-mass-accuracy mass spectrometer. It combines a quadrupole mass filter with an Orbitrap mass analyzer to provide accurate mass measurements and high-resolution separations of complex samples.

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Close spelling variants of this product

Q Exactive (1165 citations) Q Exactive HF (338 citations) Q Exactive Orbitrap (257 citations) Orbitrap Q Exactive HF-X mass spectrometer (147 citations) Q Exactive HF Orbitrap mass spectrometer (142 citations) Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (110 citations) Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (69 citations) Q Exactive HF-X Quadrupole-Orbitrap mass spectrometer (18 citations) Orbitrap Exactive (13 citations) Orbitrap Q-Exactive HF-X (13 citations)

47 protocols using q exactive hf orbitrap

High-Resolution Mass Spectrometry Analysis of Peptide Modifications

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HRMS and MS² measurements of the products of peptide extension were performed
as described previously.^{17 (link)} Samples were
analyzed using an Orbitrap QExactive Plus (MS1 at 35K resolution,
MS2 at 17.5K res; Thermo Scientific), except for meta-Bht that was
analyzed using an Orbitrap QExactive HF (MS1 @ 60K res, MS2 @ 30K
res; Thermo Scientific), all at 27% normalized collision energy (nce).
Hexapeptide products were confirmed by HRMS (Supporting Information Table 2), and the incorporation of the variable
residues at position 6 of the peptide were confirmed by monitoring
the amino acid specific ammonium ions of the peptides in MS² measurements from the hydrolyzed peptides. Conformation of the presence
of a β-hydroxyl moiety in these C-terminal residues was further
confirmed by monitoring for these amino acid specific ammonium ions
having also eliminated a water molecule (Supporting Information Table 3).

Kaniusaite M., Goode R.J., Schittenhelm R.B., Makris T.M, & Cryle M.J. (2019). The Diiron Monooxygenase CmlA from Chloramphenicol Biosynthesis Allows Reconstitution of β-Hydroxylation during Glycopeptide Antibiotic Biosynthesis. ACS Chemical Biology, 14(12), 2932-2941.

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Optimized Metabolomic Profiling of Samples

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All samples were analyzed on an Orbitrap Q Exactive HF (Thermo Scientific) coupled to Waters nanoAcquity UPLC using a previously optimized protocol [6 (link),36 (link)]. The chromatographic stationary phase was a nanoscale column (75 μm × 100 mm) packed with 1.9 μm C18 beads to facilitate the reverse-phase analysis or a SeQuant ZIC-HILIC column (2.1 mm × 150 mm, 3.5 μm resin) for chromatographic separation of polar metabolites. For the reverse phase C18 separation, mobile phases A and B each consisted of LC-MS grade water or acetonitrile, respectively, with 0.2% formic acid. The sample injection volume was 2 μL, while the system flow rate was 0.25 μL/min with a biphasic gradient in 60 min. For the HILIC analysis, 10 mM ammonium acetate in LC-MS grade water or acetonitrile with pH 8 was used for mobile phase A and B. The sample injection volume was 2 µL, while the system flow rate was 0.1 mL/min with a 90 min triphasic gradient. Full MS scans used a resolution of 120,000 from m/z 100–1000. Automatic gain control was set to 3e6 for full MS. A top 20 MS/MS method used HCD at a stepped normalized collision energy of 50, 100, and 150 for fragmentation and a resolution of 30,000 in the Orbitrap. Isolation width in the ion trap was 1 Da. Each MS/MS scan had a target of 3e5 automatic gain control. Dynamic exclusion was set at 20 s.

Wang X., Cho J.H., Poudel S., Li Y., Jones D.R., Shaw T.I., Tan H., Xie B, & Peng J. (2020). JUMPm: A Tool for Large-Scale Identification of Metabolites in Untargeted Metabolomics. Metabolites, 10(5), 190.

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Quantitative Proteomics by LC-MS/MS

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LC-MS/MS analysis was performed on a Q-Exactive HF. 5μL of total peptides were analyzed on a Waters M-Class UPLC using a C18 25cm Thermo EASY-Spray column (2um, 100A, 75um x 25cm) or IonOpticks Aurora ultimate column (1.7um, 75um x 25cm) coupled to a benchtop ThermoFisher Scientific Orbitrap Q Exactive HF mass spectrometer. Peptides were separated at a flow rate of 400 nL/min with the following gradients: 70 minutes (SEC-DIA), 160 minutes (SEC-TMT and DIA runs for non-fractionated samples), all including sample loading and column equilibration times. For DIA runs MS1 Spectra were measured with a resolution of 120,000, an AGC target of 5e^{6 (link)} and a mass range from 350 to 1650 m/z. 15 isolation windows of 87 m/z were measured at a resolution of 30,000, an AGC target of 3e^{6 (link)}, normalized collision energies of 22.5, 25, 27.5, and a fixed first mass of 200 m/z. For DDA runs MS1 Spectra were measured with a resolution of 120,000, an AGC target of 3e^{6 (link)} and a mass range from 300 to 1800 m/z. Top12 MS2 spectra were acquired at a resolution of 60,000, an AGC target of 1e^{5 (link)}, an isolation window of 0.8m/z, normalized collision energies of 28, and a fixed first mass of 110 m/z.

Doron-Mandel E., Bokor B.J., Ma Y., Street L.A., Tang L.C., Abdou A.A., Shah N.H., Rosenberger G.A, & Jovanovic M. (2023). SEC-TMT facilitates quantitative differential analysis of protein interaction networks. bioRxiv.

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Comprehensive DIA-based Plasma Proteomics

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The plasma samples from 171 patients who were enrolled in the participating centers in Bochum and Bonn were digested according to the SP3 protocol with slight modifications. Briefly, 100 µg protein was purified using paramagnetic beads (Cytiva Sera-Mag Carboxyl-Magnet-Beads, GE Healthcare, Chicago, IL) and digested overnight using trypsin (SERVA Electrophoresis, Heidelberg, Germany). Subsequently, 300 ng tryptic peptides per sample were analyzed using an Ultimate 3000 RSLCnano HPLC coupled online to either an Orbitrap QExactive, Orbitrap QExactive HF, or Orbitrap Fusion Lumos mass spectrometer (all Thermo Scientific, Bremen, Germany). In total, 306 samples were analyzed and distributed over five batches and separated by either a 96-min (Batch 1) or 38-min (Batches 2–5) LC gradient. The mass spectrometers were operated in data-independent acquisition mode. Spectral libraries were generated with FragPipe (v.17.1) and protein quantification was conducted using DIA-NN (v.1.8) [30 (link)]. The Uniprot/SwissProt database restricted to homo-sapiens (release 01_2022; 20,386 entries) was used for protein identification. The resulting protein intensities were first normalized using the LOESS method [31 (link)]. The subsequent cross-batch normalization was based on linear regression models. A detailed description of the applied methods can be found in the Additional file.

Unterberg M., Ehrentraut S.F., Bracht T., Wolf A., Haberl H., von Busch A., Rump K., Ziehe D., Bazzi M., Thon P., Sitek B., Marcus K., Bayer M., Schork K., Eisenacher M., Ellger B., Oswald D., Wappler F., Defosse J., Henzler D., Köhler T., Zarbock A., Putensen C.P., Schewe J.C., Frey U.H., Anft M., Babel N., Steinmann E., Brüggemann Y., Trilling M., Schlüter A., Nowak H., Adamzik M., Rahmel T, & Koos B. (2023). Human cytomegalovirus seropositivity is associated with reduced patient survival during sepsis. Critical Care, 27, 417.

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Mass Spectrometry Proteomics Workflow

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Resin containing purified immunoprecipitates was incubated in 200 mM HEPES (4-(hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5 containing 5 mM dithiothreitol (Sigma-Aldrich) at 37°C for 1 hr, followed by alkylation of cysteine residues using 15 mM iodoacetamide (Sigma-Aldrich) in the dark at room temperature for 1 hour. Alkylated proteins were diluted in 1:6 ratio (v/v) in ultrapure water prior to digestion using sequencing-grade trypsin (Worthington Biochemical Corp) at 37°C for 16 hrs. Digested peptides were subsequently desalted using self-packed C18 Stage Tips (3M Empore™) (Rappsilber et al., 2003) for LC-MS/MS analysis. Desalted peptides were resuspended in 0.1% (v/v) formic acid and analyzed on an Easy-nLC 1000 (Thermo Fisher Scientific) coupled to Orbitrap Q-Exactive HF (Thermo Fisher Scientific) mass spectrometer. Chromatography for peptide separation was performed using increasing organic proportion of acetonitrile (5–40 % (v/v)) on a self-packed analytical column using PicoTip™ emitter (New Objective, Woburn, MA) containing Reprosil Gold 120 C18, 1.9 um particle size resin (Dr. Maisch, Ammerbuch-Entringen, Germany) over a 120-min gradient at a flow rate of 300 nl/min. The mass spectrometry analyzer was operated in data dependent acquisition mode with a top ten method at a mass range of 300 – 2000 Da.

Chung J., Wu X., Lambert T.J., Lai Z.W., Walther T.C, & Farese RV J.r. (2019). Lipid droplet assembly factor-1 and seipin form a lipid droplet assembly complex. Developmental cell, 51(5), 551-563.e7.

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Peptide Analysis by nLC-MS/MS

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Samples were analyzed as described in^{81 (link)} with few exceptions. In particular, 4 µL of peptide mixture from each sample were analyzed on a nLC–ESI–MS/MS quadrupole Orbitrap QExactive-HF mass spectrometer (Thermo Fisher Scientific). Peptides separation was achieved on a linear gradient from 95% solvent A (2% ACN, 0.1% formic acid) to 60% solvent B (80% acetonitrile, 0.1% formic acid) over 48 min, and from 60 to 100% solvent B in 2 min at flow rate of 0.25 µL/min on a UHPLC Easy-nLC 1000 (Thermo Fischer Scientific) connected to a 23 cm fused-silica emitter of 75 µm inner diameter (New Objective, Inc. Woburn, MA, USA), packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads (Dr. Maisch GmbH, Ammerbuch, Germany). MS data were acquired using a data-dependent top 15 method for HCD fragmentation. Survey full scan MS spectra (300–1800 Th) were acquired in the Orbitrap with 60,000 resolution, AGC target 3^e6, IT 20 ms. For HCD spectra, resolution was set to 15,000 at m/z 200, AGC target 1^e^{5 (link)}, IT 160 ms; NCE 28% and isolation width 1.4 m/z.

Bruckmann C., Tamburri S., De Lorenzi V., Doti N., Monti A., Mathiasen L., Cattaneo A., Ruvo M., Bachi A, & Blasi F. (2020). Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis. Scientific Reports, 10, 16809.

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Direct Infusion Mass Spectrometry of Proteins

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A 6 μL aliquot of each 50 μM protein solution was loaded into a fresh EconoTip Emitter, 1.2 mm OD with standard coating (New Objective, Woburn, MA). Emitters were mounted on a Nanospray Flex Ion Source fitted with a NanoES off-Line kit ES259, and spectra were collected on Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Direct infusion of sample into the instrument was effected without any applied hydrostatic pressure, but solely by the spray voltage of 1.5–1.7 kV in positive ion mode. Flow rate was estimated to be 20 – 80 nL/min. Heated capillary was set at 250°C and the S-Lens RF level was 55%. Scan range was 400 – 2000 m/z for samples added to 50% MeOH with 0.05% formic acid or 400 – 5000 m/z for samples in 10 mM ammonium acetate, resolution was 60,000, AGC target 1e6, and maximum inject time was 30 ms.

Welsch M.E., Kaplan A., Chambers J.M., Stokes M.E., Bos P.H., Zask A., Zhang Y., Sanchez-Martin M., Badgley M.A., Huang C.S., Tran T.H., Akkiraju H., Brown L.M., Nandakumar R., Cremers S., Yang W.S., Tong L., Olive K.P., Ferrando A, & Stockwell B.R. (2017). Multivalent small molecule pan-RAS inhibitors. Cell, 168(5), 878-889.e29.

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Enzymatic Nucleoside Extraction and Analysis

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Nucleosides were prepared from enzyme-processed RNA by enzymatic digestion, using a cocktail of Benzonase (Merck), Phosphodiesterase 1 (Merck), and Antarctic Phosphatase (New England Biolabs) as described previously (van Delft et al., 2017 (link)). The reactions were filtered using an Amicon 30kDa MWCO spin-column (Merck) to remove protein and the filtrate was mixed with a 2x loading buffer containing 0.1% formic acid and an internal standard (¹³C-labeled uridine generated from 645672-1MG Merck KGaA, previously treated with Antarctic Phosphatase). The samples were loaded onto an ACQUITY UPLC HSS T3 Column, 100 Å, 1.8 μm, 1 mm X 100 mm (Waters Corp., Milford, MA, USA) and resolved using a gradient of 2%–10% acetonitrile in 0.1% formic acid over 10 min. Mass spectrometric analysis was performed in positive ion mode on an Orbitrap QExactive HF (Thermo Fisher, Waltham, MA, USA) mass spectrometer. Standard dilutions of all experimental nucleosides were prepared and analyzed in parallel. There were three technical replicates of each sample and the analytical processing was performed using XCalibur Software (Thermo Fisher).

Pandolfini L., Barbieri I., Bannister A.J., Hendrick A., Andrews B., Webster N., Murat P., Mach P., Brandi R., Robson S.C., Migliori V., Alendar A., d’Onofrio M., Balasubramanian S, & Kouzarides T. (2019). METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation. Molecular Cell, 74(6), 1278-1290.e9.

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Peptide Separation and Identification via LC-MS/MS

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First, 2 µg of peptides was injected onto a 0.3 × 5 mm trap-column (5 µm C18 particles, Dionex) from where they were eluted to an in-house-packed (3 µm C18 particles, Dr. Maisch) analytical 50 cm × 75 µm emitter column (New Objective). Both columns were operated at 40 °C. The peptides were separated at 250 nL/min with an 8–35% A-to-B 120 min gradient. Eluent B was 80% acetonitrile +0.1% formic acid and eluent A was 0.1% formic acid in water. The eluted peptides were sprayed into a quadrupole–Orbitrap Q Exactive HF (Thermo Fisher Scientific) MS/MS using a nano-electrospray source and a spray voltage of 2.5 kV (liquid junction connection). The MS instrument was operated with a top-12 data-dependent acquisition strategy. One 350–1400 m/z MS scan (at a resolution setting of 60,000 at 200 m/z) was followed by an MS/MS (R = 30 000 at 200 m/z) of the 12 most intense ions using higher-energy collisional dissociation fragmentation (normalized collision energy of 26). The MS and MS/MS ion target and injection time values were 3 × 10⁶ (50 ms) and 1 × 10⁵ (41 ms), respectively. The dynamic exclusion time was limited to 45 s; only charge states +2 to +6 were subjected to MS/MS.

Saar M., Jaal J., Meltsov A., Laasfeld T., Lust H., Kasvandik S, & Lavogina D. (2023). Exploring the Molecular Players behind the Potentiation of Chemotherapy Effects by Durvalumab in Lung Adenocarcinoma Cell Lines. Pharmaceutics, 15(5), 1485.

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LC-MS/MS analysis of pre-fractionated samples

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The LC–MS/MS analysis of pre-fractionated samples was performed using UltiMate 3000 RSLCnano system coupled with a Thermo Scientific Orbitrap Q Exactive HF. Mobile phase A consisted of 0.2% formic acid (FA), 3% DMSO in water and mobile phase B consisted of 0.2% FA, 3% DMSO in 67% acetonitrile. Peptides were loaded into a CoAnn C18 column (75 μm × 20 cm, 1.9 μm particles) at a flow rate of 250 nL/min with the following gradient: 12–22% mobile phase B over 30 min, 22–40% mobile phase B in 30 min, 40–60% mobile phase B in 7 min, 60–95% mobile phase B in 3 min, stay at 95% mobile phase B for 3 min, followed by 15 min equilibration (44 (link)).
MS data were acquired using a ‘high-high’ acquisition method (high resolution on both MS1 and MS2). MS1 scans were detected in the Orbitrap at 120K resolution in the m/z range 400–2000 and AGC target of 1 × 10⁶ with a maximum injection time of 50 ms. Ions with charge states from 2+ to 8+ were selected for fragmentation by stepped high energy collision dissociation (HCD) at 27%, 30% 33% with AGC of 1 × 10⁵, maximum injection time of 100 ms, and detected in the Orbitrap at 60K resolution.

Bwayi M.N., Garcia-Maldonado E., Chai S.C., Xie B., Chodankar S., Huber A.D., Wu J., Annu K., Wright W.C., Lee H.M., Seetharaman J., Wang J., Buchman C.D., Peng J, & Chen T. (2022). Molecular basis of crosstalk in nuclear receptors: heterodimerization between PXR and CAR and the implication in gene regulation. Nucleic Acids Research, 50(6), 3254-3275.

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