Orbitrap id x tribrid mass spectrometer

Manufactured by Thermo Fisher Scientific

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The Orbitrap ID-X Tribrid mass spectrometer is a high-performance analytical instrument designed for advanced proteomics and metabolomics research. It combines an Orbitrap mass analyzer with additional ion traps, enabling high-resolution, accurate-mass measurements and tandem mass spectrometry capabilities.

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Orbitrap mass spectrometers (4 citations) Orbitrap ID-X Tribrid (3 citations) ID-X mass spectrometer (2 citations) Orbitrap ID-X (2 citations)

11 protocols using orbitrap id x tribrid mass spectrometer

Metabolomic Profiling by Tandem Mass Spectrometry

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MS/MS spectra for polar metabolites were acquired on an Orbitrap ID-X Tribrid mass spectrometer (Thermo Scientific). A Vanquish Horizon UHPLC system, with the same chromatographic conditions as described in the Methods, was interfaced with the mass spectrometer via electrospray ionization in both positive and negative mode with a spray voltage of 3.5 and 2.8 kV, respectively. The RF lens value was 35%. Data were acquired in data dependent acquisition (DDA) mode using the built-in deep scan option (AcquireX) with a mass range of 67–900 m/z. MS/MS scans were acquired at 15K resolution on a NIST SRM 1950 plasma sample from and 4 individual samples from d0, d3, d7, and d14 in both positive and negative polarity with different collision energies in the range of 20 NCE to 50 NCE for HCD and 30 NCE for CID to maximize identifications.
MS/MS spectra for polar metabolites and lipids were acquired using an iterative approach in the MassHunter Acquisition Software (Version 10.1.48, Agilent Technologies) on an Agilent 6540 and 6545 QTOF respectively. The same source settings as for MS1 data acquisition were used. MS/MS spectra were acquired at a scan rate of 3 spectra/s with different intensity thresholds and collision energies of 10, 20, and 40 V to increase identification rates.

Sindelar M., Stancliffe E., Schwaiger-Haber M., Anbukumar D.S., Albrecht R.A., Liu W.C., Travis K.A., García-Sastre A., Shriver L.P, & Patti G.J. (2021). Longitudinal Metabolomics of Human Plasma Reveals Robust Prognostic Markers of COVID-19 Disease Severity. medRxiv.

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Lipid Profiling by RP-UHPLC-MS

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Reverse-phase (RP) ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis was performed with a Thermo Accucore C30, 150 × 2.1 mm, 2.6 μm particle size column mounted in a Vanquish LC coupled to an Orbitrap ID-X Tribrid mass spectrometer (ThermoFisher Scientific). The mobile phases and chromatographic gradients used are described in Supplementary Table S6. MS data were acquired in positive and negative ion modes in the 150–2000 m/z range with a 120,000 mass resolution setting. The most relevant MS parameters are provided in the supplementary section Table S7. Samples were kept at 4 °C in the autosampler during LC-MS analysis while the column temperature was set to 50 °C. An injection volume of 2 μL was used for all runs. For lipid annotation, MS/MS experiments were performed using the Thermo Scientific AcquireX data acquisition workflow. Tandem MS data were acquired at a resolution of 30,000 and an isolation window of 0.4 m/z. Precursor ions were fragmented with HCD and CID activation methods. For HCD, stepped normalized collision energy (NCE) of 15, 30, and 45 and a CID collision energy of 40 were used to fragment the precursor ions.

Bifarin O.O., Sah S., Gaul D.A., Moore S.G., Chen R., Palaniappan M., Kim J., Matzuk M.M, & Fernández F.M. (2023). Machine Learning Reveals Lipidome Remodeling Dynamics in a Mouse Model of Ovarian Cancer. bioRxiv.

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Quantifying Unsaturated Fatty Acids in Tumor-Bearing Mice

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Serum was collected from N=8 mice bearing TC-1 tumors (4 with HFD and 4 with ND) and the level of unsaturated free fatty acids (uFFAs) was determined by mass spectrometry. Briefly, to extract metabolites from serum, samples were diluted 1:9 with methanol:acetonitrile:water, vortexed for 1 min, and incubated at −20°C for 1 h. Following incubation, metabolite extracts were centrifuged at 14K rpm at 4°C for 10 min, and the supernatant was transferred into an LC/MS vial for LC/MS analysis. Ultra-high performance LC (UHPLC)/MS was performed with a Thermo Scientific Vanquish Horizon UHPLC system interfaced with a Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer (Thermo Scientific, Waltham, MA). Chromatographic separation was accomplished by using a Waters Acquity UPLC T3 column (150 mm × 2.1 mm, 1.8 μm). LC/MS data were processed and analyzed with the open-source Skyline software(26 (link)). Results are reported as a relative normalized intensity for each fatty acid for HFD mice (N=4) versus the averaged value from N= 4 ND mice as a control.

Muhammad N., Ruiz F., Stanley J., Rashmi R., Cho K., Jayachandran K., Zahner M.C., Huang Y., Zhang J., Markovina S., Patti G.J, & Schwarz J.K. (2022). Mono- and diunsaturated fatty acids sensitize cervical cancer to radiation therapy. Cancer research, 82(24), 4515-4527.

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Optimized MS/MS Acquisition for Metabolite Identification

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MS/MS spectra for polar metabolites and lipids were acquired by using an iterative approach in the MassHunter Acquisition Software (Version 10.1.48, Agilent Technologies) on an Agilent 6540 and 6545 QTOF, respectively. The same source settings as for MS1 data acquisition were used. MS/MS spectra were acquired at a scan rate of 3 spectra/s with different intensity thresholds and collision energies of 10, 20, and 40 V to increase identification rates.
To improve matching to Orbitrap spectral databases, MS/MS data for polar metabolites were acquired on an Orbitrap ID-X Tribrid mass spectrometer (Thermo Scientific). A Vanquish Horizon UHPLC system, with the same chromatographic conditions as described in the Method details, was interfaced with the mass spectrometer via electrospray ionization in both positive and negative ion mode with a spray voltage of 3.5 and 2.8 kV, respectively. The RF lens value was 35%. Data were acquired in data dependent acquisition (DDA) mode by using the built-in deep scan option (AcquireX) with a mass range of 67-900 m/z. MS/MS scans were acquired at 15K resolution on a NIST SRM 1950 plasma sample and from 4 individual samples (d0, d3, d7, and d14) in both positive and negative ion mode with different collision energies in the range of 20 NCE to 50 NCE for HCD and 30 NCE for CID to maximize identifications.

Sindelar M., Stancliffe E., Schwaiger-Haber M., Anbukumar D.S., Adkins-Travis K., Goss C.W., O’Halloran J.A., Mudd P.A., Liu W.C., Albrecht R.A., García-Sastre A., Shriver L.P, & Patti G.J. (2021). Longitudinal metabolomics of human plasma reveals prognostic markers of COVID-19 disease severity. Cell Reports Medicine, 2(8), 100369.

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RP-UHPLC-MS Lipid Profiling Protocol

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Reverse-phase (RP) ultra-high
performance liquid chromatography–mass spectrometry (UHPLC–MS)
analysis was performed with a Thermo Accucore C30, 150 × 2.1
mm², 2.6 μm particle size column mounted in a Vanquish
LC coupled to an Orbitrap ID-X Tribrid mass spectrometer (ThermoFisher
Scientific). The mobile phases and chromatographic gradients used
are described in Table S6. MS data were
acquired in positive and negative ion modes in the 150–2000 m/z range with a 120,000 mass resolution
setting. The most relevant MS parameters are provided in Table S7. Samples were kept at 4 °C in the
autosampler during LC–MS analysis, while the column temperature
was set to 50 °C. An injection volume of 2 μL was used
for all runs. For lipid annotation, MS/MS experiments were performed
using the Thermo Scientific AcquireX data acquisition workflow. Tandem
MS data were acquired at a resolution of 30,000 and an isolation window
of 0.4 m/z. Precursor ions were
fragmented with HCD and CID activation methods. For HCD, stepped normalized
collision energy (NCE) of 15, 30, and 45 and a CID collision energy
of 40 were used to fragment the precursor ions.

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Quantitative Metabolomics by UHPLC-Orbitrap MS

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A Vanquish UHPLC system was coupled to an Orbitrap ID-X Tribrid mass spectrometer (Thermo Fisher Scientific) via electrospray ionization with the following source conditions: sheath gas flow 50 arbitrary units (Arb), auxiliary gas flow 10 Arb, sweep gas flow 1 Arb, ion transfer tube temperature 300 °C, and vaporizer temperature 200 °C. The RF lens value was 60%. Data were acquired in negative and positive polarity with a spray voltage of 2.8 kV and 3.5 kV, respectively. MS1 data were acquired from 67 to 900 m/z at a resolution of 120,000 with an automatic gain control (AGC) target of 2e5 and a maximum injection time of 200 ms in polarity switching mode. MS2 data for metabolite identification were acquired at a resolution of 15,000 with an AGC target of 2.5e4 and a maximum injection time of 70 ms in negative and positive mode separately. A 5 ppm mass error and 10 s dynamic exclusion were applied.

Schwaiger-Haber M., Stancliffe E., Anbukumar D.S., Sells B., Yi J., Cho K., Adkins-Travis K., Chheda M.G., Shriver L.P, & Patti G.J. (2023). Using mass spectrometry imaging to map fluxes quantitatively in the tumor ecosystem. Nature Communications, 14, 2876.

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Comprehensive Metabolomic Profiling by RP-UHPLC-MS and HILIC-UHPLC-MS

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Reverse phase (RP) ultra-high-performance liquid chromatography, mass spectrometry (UHPLC-MS) and hydrophilic interaction liquid chromatography (HILIC) UHPLC-MS analysis were performed to obtain a deeper coverage of the metabolome. Serum samples were thawed on ice, followed by metabolite extraction of both non-polar (lipid) and polar metabolites using two different sample preparation protocols, detailed in the supplementary information. Reverse phase chromatography was performed with a Thermo Accucore C30, 150 × 2.1 mm, 2.6 µm particle size column, and hydrophilic interaction liquid chromatography (HILIC) chromatography was performed with a Waters ACQUITY UHPLC BEH Amide 150 × 2.1 mm, 1.7 µm particle size column. A Q Exactive HF Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) was used for RP UHPLC-MS analysis, and an Orbitrap ID-X Tribrid mass spectrometer (ThermoFisher Scientific) was used for HILIC UHPLC-MS analysis. Samples were kept at 4 °C in the autosampler during runs, and injection volumes of 2 µL and 1 µL were used for RP and HILIC methods, respectively. Chromatographic gradients, relevant MS parameters and details on MS/MS experiments for metabolite annotation are described in the Supplementary Information (Tables S2 and S3).

Sah S., Ma X., Botros A., Gaul D.A., Yun S.R., Park E.Y., Kim O., Moore S.G., Kim J, & Fernández F.M. (2022). Space- and Time-Resolved Metabolomics of a High-Grade Serous Ovarian Cancer Mouse Model. Cancers, 14(9), 2262.

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Negative-ion ESI-MS analysis of purified samples

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Negative-ion ESI-MS
was performed on an Orbitrap ID-X Tribrid mass spectrometer (Thermo
Fisher Scientific) equipped with a Vanquish UHPLC system (Thermo Fisher
Scientific). The purified samples were dissolved in CH₃CN/H₂O (1:1) at a concentration of 1 μg/μL,
of which 2 μL was injected for MSⁿ. CH₃CN/H₂O (1:1) was used as the mobile phase, with a flow
rate of 0.2 mL/min. The spray voltage was at 3.0 kV with a source
temperature of 400 °C, ion transfer tube temperature 300 °C,
RF S-lens 50 V, and sheath velocity 40 psi. Higher-energy collisional
dissociation was used for the MSⁿ. For optimal fragmentation,
normalized collision energy was adjusted to 15–30% . Precursor
selection for product-ion scanning was made manually using the Xcalibur
software Version 4.2 data system.

Cao C., Cheng Y., Zheng Y., Huang B., Guo Z., Yu L., Mulloy B., Tajadura-Ortega V., Chai W., Yan J, & Liang X. (2024). Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn. Analytical Chemistry, 96(16), 6170-6179.

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Untargeted Lipidomics Using UHPLC-Orbitrap

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Liquid chromatography separation was done with Waters BEH C8 column (2.1 mm × 100 mm, 1.7 um) using16 min gradient. Mobile phase A is 10 mM ammonium acetate with 5% methanol, 0.1% acetic acid in H₂O and mobile phase B is 0.1% acetic acid in methanol. Flowrate was set at 0.4 mL/min and column oven temperature at 30 °C. 5 µL of samples were injected into Vanquish UHPLC (ThermoScientific) connected with Orbitrap ID-X Tribrid Mass Spectrometer (ThermoScientific). For global untargeted lipidomics, AcquireX DeepScan was applied and data was acquired in both negative and positive ion mode. The MS resolution was set at 60,000 for both MS1 and MS2 scans with scan range of 200–1100 m/z. The capillary voltage in the positive mode was 3.4 kV and the negative mode was 2.4 kV, respectively. The ion transfer tube temperature is 325°C, Database search was performed by Compound Discoverer 3.3 with mzVault (MONA, GNPS, and NIST), mzCloud, and LipidBlast.

Lee J., Dimitry J.M., Song J.H., Son M., Sheehan P.W., King M.W., Travis Tabor G., Goo Y.A., Lazar M.A., Petrucelli L, & Musiek E.S. (2023). Microglial REV-ERBα regulates inflammation and lipid droplet formation to drive tauopathy in male mice. Nature Communications, 14, 5197.

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Untargeted Metabolomics Sample Preparation

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Filtrates were prepared as described for previous experiments, then applied to CHROMABOND reversed phase solid-phase extraction columns (Macherey-Nagel) under an anoxic atmosphere, after the columns had first been equilibrated with liquid chromatography–mass spectrometry (LCMS)-grade methanol followed by anoxic ultrapure water. Columns were then quickly washed with water followed by elution of samples in methanol by gravity flow. Methanol samples were stored in combusted borosilicate glass vials with an N₂ headspace and stored at −80°C before mass spectrometry. In preparation for mass spectrometry, samples were then evaporated under a flow of N₂ and then resuspended in 50 μL 70% acetonitrile. Samples were then run on the Orbitrap ID-X Tribrid Mass Spectrometer (Thermo Fisher Scientific) at the Harvard Center for Mass Spectrometry.
The data were processed and analyzed with Compound Discoverer (Thermo Scientific, version 3.3). Peaks were extracted from MS1 data, and various adducts of the same compound were grouped together, followed by retention time alignment and gap filling between samples. The data were then median centered for normalization (i.e. the medians of all compound areas in a sample are centered around the median of all samples).

Baker I.R., Matzen S.L., Schuler C.J., Toner B.M, & Girguis P.R. (2023). Aerobic iron-oxidizing bacteria secrete metabolites that markedly impede abiotic iron oxidation. PNAS Nexus, 2(12), pgad421.

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