Orbitrap fusion

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom

The Orbitrap Fusion is a high-resolution mass spectrometer designed for advanced analytical applications. It utilizes Orbitrap technology to provide accurate mass measurement and high-resolution capabilities for the identification and characterization of complex molecular samples.

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307 protocols using orbitrap fusion

Multinotch-MS3 Quantification Pipeline

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An Orbitrap Fusion (Thermo Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) were used to acquire the data. For superior quantitation accuracy, we used multinotch-MS3 ^{59 (link)}. Two microliters of each fraction was resolved on a nanocapillary reverse-phase column (75 μm internal diameter by 50 cm; PepMap RSLC C18 column, Thermo Fisher Scientific) at a flowrate of 300 nL/min using 0.1% formic acid/acetonitrile gradient system (2 to 22% acetonitrile in 110 min; 22 to 40% acetonitrile in 25 min; 6-min wash at 90% acetonitrile; 25 min re-equilibration) and directly sprayed onto the Orbitrap Fusion using EasySpray source (Thermo Fisher Scientific). The mass spectrometer was set to collect one MS1 scan [Orbitrap; 120,000 resolution; AGC target 2 × 105; max IT (maximum ionization time) 50 ms] and data-dependent, “Top Speed” (3 s) MS2 scans [collision-induced dissociation; ion trap; NCE (normalized collision energy) 35; AGC (automatic gain control) 5 × 103; max IT 100 ms]. For multinotch-MS3, the top 10 precursors from each MS2 were fragmented by high energy collisional dissociation (HCD), followed by Orbitrap analysis (NCE 55; 60,000 resolution; AGC 5 × 104; max IT 120 ms, 100 to 500 mass/charge ratio scan range).

Lefevre T.J., Wei W., Mukhaleva E., Venkata S.P., Chandan N.R., Abraham S., Li Y., Dessauer C.W., Vaidehi N, & Smrcka A.V. (2023). Stabilization of Interdomain Interactions in G protein αi Subunits Determines Gαi Subtype Signaling Specificity. bioRxiv.

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High-Sensitivity Orbitrap Fusion Proteomics

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An Orbitrap Fusion (Thermo Fisher Scientific) and RSLC Ultimate 3000 nano-UPLC (Dionex) were used to acquire the data. For superior quantitation accuracy, we used multinotch-MS3 (57 (link)). Two microliters of each fraction was resolved on a nanocapillary reverse-phase column (75 μm internal diameter by 50 cm; PepMap RSLC C18 column, Thermo Fisher Scientific) at a flow rate of 300 nl/min using 0.1% formic acid/acetonitrile gradient system (2–22% acetonitrile in 110 min; 22–40% acetonitrile in 25 min; 6-min wash at 90% acetonitrile; and 25 min reequilibration) and directly sprayed onto the Orbitrap Fusion using EasySpray source (Thermo Fisher Scientific). The mass spectrometer was set to collect one MS1 scan [Orbitrap; 120,000 resolution; automatic gain control (AGC) target 2 × 105; max IT (maximum ionization time) 50 ms] and data-dependent, “Top Speed” (3 s) MS2 scans [collision-induced dissociation; ion trap; normalized collision energy 35; AGC 5 × 103; max IT 100 ms]. For multinotch-MS3, the top 10 precursors from each MS2 were fragmented by high energy collisional dissociation, followed by Orbitrap analysis (normalized collision energy 55; 60,000 resolution; AGC 5 × 104; max IT 120 ms, 100–500 mass/charge ratio scan range).

Lefevre T.J., Wei W., Mukhaleva E., Meda Venkata S.P., Chandan N.R., Abraham S., Li Y., Dessauer C.W., Vaidehi N, & Smrcka A.V. (2024). Stabilization of interdomain interactions in G protein α subunits as a determinant of Gαi subtype signaling specificity. The Journal of Biological Chemistry, 300(5), 107211.

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DYRK1B Prolyl Hydroxylation Identification

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To identify the sites of DYRK1B prolyl hydroxylation, U87 cells expressing FLAG tagged DYRK1B in the presence or the absence of PHD1 were used in immunoprecipitation assay performed with anti-hydroxyl-proline followed by anti-FLAG immunoprecipitation. Eluates containing DYRK1B protein were reduced with 5 mM DTT and alkylated with 10 mM iodoacetamide. Trypsin was added and samples were incubated at 37°C for 16 hours. 10% of the digested samples were injected into the LC/MS system comprising the Dionex Ultimate 3000 RSLCnano system and Orbitrap Fusion (ThermoFisher Scientific). Samples were loaded onto the Acclaim PepMap C18 pre-column (2 cm x 75 μm, ThermoFisher Scientific) with the loading pump at 3 μl/min for 3 min. A valve was switched to bring the pre-column into the flow path with the analytical column (Resprosil-C18, 2.4 μm, 25 cm x 75 μm, Dr. Maisch GmbH) and a gradient from 2% buffer A (0.1% formic acid in water) to 20% buffer B (acetonitrile-0.1% formic acid) in 20 min to 30% buffer B in 2 min and 80% buffer B in 1 min at 200 nl/min. Data were acquired on an Orbitrap Fusion (ThermoFisher Scientific) which detects MS/MS of individual peptides. These included the oxidized and non-oxidized peptides as well as control peptides from DYRK1B.

Lee S.B., Ko A., Oh Y.T., Shi P., D’Angelo F., Frangaj B., Koller A., Chen E.I., Cardozo T., lavarone A, & Lasorella A. (2020). Proline hydroxylation primes protein kinases for autophosphorylation and activation. Molecular cell, 79(3), 376-389.e8.

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Orbitrap Fusion Multinotch-MS3 Quantification

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For data acquisition, an Orbitrap Fusion (ThermoFisher) and RSLC Ultimate 3000 nano-UPLC (Dionex) was used to obtain raw data. To increase accuracy and confidence in protein abundance measurements, a multinotch-MS3 method was employed for MS data analysis. Two microliters from each fraction were resolved in 2D on a nanocapillary reverse phase column (Acclaim PepMap C18, 2 micron, 75 μm i.d. × 50 cm, ThermoFisher) using a 0.1% formic/acetonitrile gradient at 300 nl/m (2–22% acetonitrile in 150 m, 22–32% acetonitrile in 40 m, 20 min wash at 90% followed by 50 min reequilibration) and directly sprayed onto Orbitrap Fusion with EasySpray (ThermoFisher; Spray voltage (positive ion) = 1900 V, Spray voltage (negative ion) = 600 V, method duration = 180 min, ion source type = NSI). The mass spectrometer was set to collect the MS1 scan (Orbitrap; 120 K resolution; AGC target 2 × 10⁵; max IT 100 ms), and then data-dependent Top Speed (3 s) MS2 scans (collision induced dissociation; ion trap; NCD 35; AGC 5 × 10³; max IT 100 ms). For multinotch-MS3, the top 10 precursor ions from each MS2 scan were fragmented by HCD followed by Orbitrap analysis (NCE 55; 60 K resolution; AGC 5 × 10⁴; max IT 120 ms; 100-500 m/z scan range).

Djomehri S.I., Gonzalez M.E., da Veiga Leprevost F., Tekula S.R., Chang H.Y., White M.J., Cimino-Mathews A., Burman B., Basrur V., Argani P., Nesvizhskii A.I, & Kleer C.G. (2020). Quantitative proteomic landscape of metaplastic breast carcinoma pathological subtypes and their relationship to triple-negative tumors. Nature Communications, 11, 1723.

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UPLC-HR-MS/MS Analysis of Ethanol Extracts

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Ultra-high-performance liquid chromatography (UPLC) high-resolution tandem mass spectrometry (HR-MS/MS) analysis were carried out using an Orbitrap Fusion (Thermo Electron Co., Waltham, MA, USA) coupled to an Acquity UPLC system (Waters, Milford, MA, USA). Chromatographic separation of ethanol extract was conducted using a Acquity UPLC^® BEH C18 column (“2.1 × 150 mm”, 1.7 μm), operated at 50 °C and using mobile phases A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid). The flow rate and injection volumes were 0.4 mL/min and 3 µL, respectively. The elution gradients were as follows: 0–1 min, 5% B (isocratic); 1–20 min, 5–70% B (linear gradient); 20–24 min, 70–100% B (linear gradient); 24–26 min, 100% B (isocratic). The LC-MS system consisted of heated electrospray ionization (HESI) probe as the ionization source. MS analysis was performed with negative ion mode and the following parameters for MS/MS scan: m/z range of 100–1800; collision-induced dissociation energy of 45%; data-dependent scan mode. The Orbitrap analyzer was used for high-resolution mass spectra data acquisition with a mass resolving power of 60,000 full width at half maximum (FWHM).

Cho J.H., Kim D.H., Lee J.S., Seo M.S., Kim M.E, & Lee J.S. (2022). Sargassum horneri (Turner) C. Agardh Extract Regulates Neuroinflammation In Vitro and In Vivo. Current Issues in Molecular Biology, 44(11), 5416-5426.

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Orbitrap-based LC-MS/MS Analysis of Peptides

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LC-MS/MS data acquisition was carried out on an Orbitrap Fusion mass spectrometer (Thermo Electron, Bremen, Germany), interfaced with Easy-nLC1000 nanoflow LC system (Thermo Scientific, Odense, Denmark). All fractions were analyzed into technical triplicates. The peptides were later reconstituted in 0.1 % formic acid and loaded onto a trap column (nanoviper 2 cm, 3 µm magic C18Aq, Thermo Scientific). Peptides were resolved on an analytical column (nanoviper 25 cm (75 µm silica capillary, 3 µm magic C18, Thermo Scientific)), at a flow rate of 300 nL/min, using a linear gradient of 2–38 % solvent B (0.1 % formic acid in 100 % ACN) for 100 min. The total run time was 120 min. Data-dependent acquisition with full scans in 350–1600 m/z range was performed using an Orbitrap mass analyzer at a mass resolution of 120,000 at 400 m/z. The most intense precursor ions (top 20) from a survey scan were selected for MS/MS fragmentation using higher energy collision dissociation (HCD) fragmentation, with 34 % normalized collision energy and detected at a mass resolution of 50,000. AGC target value was set to 1,000,000 with maximum ion injection time of 150 ms.

Mohanraj N., Joshi N.S., Poulose R., Patil R.R., Santhoshkumar R., Kumar A., Waghmare G.P., Saha A.K., Haider S.Z., Markandeya Y.S., Dey G., Rao L.T., Govindaraj P, & Mehta B. (2022). A proteomic study to unveil lead toxicity-induced memory impairments invoked by synaptic dysregulation. Toxicology Reports, 9, 1501-1513.

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High-Resolution Orbitrap-based Proteomics

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NanoLC-MS/MS analyses were performed on an Orbitrap Fusion (Thermo Scientific) equipped with a PicoView Ion Source (New Objective) and coupled to an EASY-nLC 1000 (Thermo Scientific). Peptides were loaded on a trapping column (2 cm x 150 μm ID, PepSep) and separated on capillary columns (30 cm × 150 μm ID, PepSep) that were both packed with 1.9 μmC18 ReproSil and separated with a 45 min linear gradient from 3% to 30% acetonitrile and 0.1% formic acid with a flow rate of 500 nl/min. MS, MS2, and MS3 scans were acquired in the Orbitrap analyzer with a resolution of 60,000. For MS2 spectra, HCD fragmentation with a stepped collision energy of 35%, 40%, 45% was applied. For MS3 spectra, HCD fragmentation with a collision energy of 55% was applied. A top speed, data-dependent MS/MS method with a fixed cycle time of 3 s was used. Dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 30 s. Singly charged precursors were excluded from selection. The minimum signal threshold for precursor selection was set to 50,000. Standard AGC targets were set for MS and MS2 scans. For the MS3 scans, a customized AGC target of 300% was set. EASY-IC was used for internal calibration.

Mohnke J., Stark I., Fischer M., Fischer P.M., Schlosser A., Grothey A., O’Hare P., Sodeik B., Erhard F., Dölken L, & Hennig T. (2022). pUL36 Deubiquitinase Activity Augments Both the Initiation and the Progression of Lytic Herpes Simplex Virus Infection in IFN-Primed Cells. Journal of Virology, 96(22), e00963-22.

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Quantitative Proteomics via LC-MS/MS

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LC-MS analysis was performed, modified from (66 (link)) using an Easy-nLC 1000 nanoLC chromatography system interfaced to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Samples were preconcentrated and desalted on to a C18 trap column prior to separation over a 180-min gradient from 0% to 50% mobile phase B (A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile) at 300 nl/min flow rate with a 75 μm × 15 cm PepMap rapid separation liquid chromatography column (Thermo Fisher Scientific). MS parameters were as follows: ion spray voltage 2000V, survey scan MS1 120k resolution with a 2 s cycle time, interleaved with data-dependent ion trap MS/MS of highest intensity ions with charge state-dependent ion selection window (z = 2:1.2 m/z, z = 3:0.7 m/z, z = 4–6:0.5 m/z) and CID at 35% normalized collision energy. Additionally, the top five (z = 2) or 10 (z > 2) product ions were synchronously selected for higher collisional dissociation MS³ at normalized collision energy 65% with Orbitrap detection to generate TMT-tag intensities.

Canniff N.P., Graham J.B., Guay K.P., Lubicki D.A., Eyles S.J., Rauch J.N, & Hebert D.N. (2023). TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein. The Journal of Biological Chemistry, 299(12), 105450.

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Nanoflow LC-MS/MS Peptide Analysis

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All samples were analyzed on a Thermo Scientific Orbitrap Fusion Tribrid (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Ultimate 3000 Nano UHPLC system (Dionex, Sunnyvale, CA, USA) using a 150 × 0.075 mm PepMap 100 C18, 2 μm particle size, analytical column (Thermo Fisher Scientific) in trapping mode with a C18 trap cartridge. The peptides were separated using mobile phase A (water with 0.1% formic acid) and mobile phase B (acetonitrile with 0.1% formic acid) at a flow rate of 0.3 μL/min. The peptide separation gradient consisted of 2 to 30% solvent B over 4 min, ramped to 95% solvent B over 2 min, held for 4 min, and then returned to 2% solvent B over 1 min and held for 6 min. Peptides were eluted directly into the nanospray source of an Orbitrap Fusion instrument controlled with Xcalibur version 3.1 (Thermo Fisher, San Jose, CA) using a conductive nanospray emitter. The spray voltage was set to 2400 V and ion transfer tube was set to 300 °C. The MS1 data were collected at a resolution of 60000 with the scan range of 250–2000 m/z. In CID mode, full MS scans were followed by eight subsequent MS/MS scans on the top eight most abundant peptide ions. Collision energy was set to 35%.

Mitra S., Talukdar K., Prasad P., Misra S.K., Khan S., Sharp J.S., Jurss J.W, & Chakraborty S. (2021). Rational Design of a Cu Chelator that Mitigates Cu-Induced ROS Production by Amyloid Beta. Chembiochem : a European journal of chemical biology, 23(4), e202100485.

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Orbitrap-based LC-MS/MS Proteomics Analysis

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LC–MS/MS analysis was conducted using an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific) and an Orbitrap Fusion (Thermo Fisher Scientific). The sample was reconstituted using buffer A (0.1% formic acid, 2% ACN) and then loaded onto C18 reversed phase capillary analytical column (3 μm particle size, 90 Å pore size) by the autosampler which connected to an EASY-nLC 1000 HPLC system. The peptides were eluted at constant flow rate of 300 nL/min by increasing buffer B (0.1% formic acid, 90% ACN) from 8% to 32% over 58 min, then 48% in 6 min followed a steep increase to 80% in 2 min.
The mass spectrometric analysis was carried out in a data dependent acquisition (DDA) mode with a cycle time of 3 s. The peptides with a range of m/z 350–1300 were detected by an Orbitrap mass analyzer. And the resolution was set as 120,000 at m/z 200. Automatic gain control (AGC) target and maximum ion injection time (IT) were 5.0 × 105 and 50 ms, respectively. The isolated precursor ions were subjected to fragmentation via higher-energy collisional dissociation (HCD) with a collision energy of 32%, and analyzed by ion trap analyzer. The dynamic exclusion was set as 60 s, and the charge inclusion state was set to 2 – 6 + .

Wang R., Xiao L., Pan J., Bao G., Zhu Y., Zhu D., Wang J., Pei C., Ma Q., Fu X., Wang Z., Zhu M., Wang G., Gong L., Tong Q., Jiang M., Hu J., He M., Wang Y., Li T., Liang C., Li W., Xia C., Li Z., Ma D.K., Tan M., Liu J.Y., Jiang W., Luo C., Yu B, & Dang Y. (2023). Natural product P57 induces hypothermia through targeting pyridoxal kinase. Nature Communications, 14, 5984.

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