Q exactive quadrupole orbitrap instrument

Manufactured by Thermo Fisher Scientific

The Q-Exactive Quadrupole-Orbitrap instrument is a high-resolution mass spectrometer designed for accurate mass measurement and high-quality data acquisition. It combines a quadrupole mass filter with an Orbitrap mass analyzer, enabling precise mass analysis and tandem mass spectrometry capabilities.

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Lab products found in correlation

Ultimate 3000 nano system, by Thermo Fisher Scientific (3 mentions) Mascot server, by Matrix Science (2 mentions) Progenesis qi software, by Waters Corporation (2 mentions) Strataclean resin, by Agilent Technologies (1 mentions) Easy spray analytical column, by Thermo Fisher Scientific (1 mentions) Nanoacquity ultra performance lc, by Waters Corporation (1 mentions)

Spelling variants of this product

Q Exactive (1165 citations) Q Exactive Orbitrap (257 citations) Q Exactive instrument (48 citations) Orbitrap Exactive (13 citations) Q Exactive Quadrupole-Orbitrap (8 citations) Q-Exactive Orbitrap instrument (5 citations) Orbitrap Exactive instrument (5 citations)

7 protocols using q exactive quadrupole orbitrap instrument

LC-MS/MS Proteomics Workflow

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All digests were individually
analyzed via LC-MS/MS on an UltiMate 3000 Nano LC System (Dionex/Thermo
Scientific) coupled to a Q Exactive Quadrupole-Orbitrap instrument
(Thermo Scientific). Full LC-MS/MS instrument methods are described
in the Supporting Information. Tryptic
peptides (equivalent to 200 ng of protein) were loaded onto the column
and run over a 30, 60, 90, or 120 min LC gradient as stated.

Anderson J.R., Phelan M.M., Rubio-Martinez L.M., Fitzgerald M.M., Jones S.W., Clegg P.D, & Peffers M.J. (2020). Optimization of Synovial Fluid Collection and Processing for NMR Metabolomics and LC-MS/MS Proteomics. Journal of Proteome Research, 19(7), 2585-2597.

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Proteomic Analysis of U3 snoRNA Knockdown

Cited 4 times

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Cell lysates following U3 snoRNA knockdown (or control) in SW1353 cells were used^{21 (link)}. Cells were harvested from triplicate wells of a six well plate with parallel wells used for confirmation of U3 snoRNA knockdown. In-solution tryptic digestion on 10 μg protein was undertaken as previously described^{22 (link)}. For the secretomes in-solution tryptic digestion was carried out on 10 µl of StrataClean resins (Agilent Genomics, 400,714) on 100 µg of protein for each sample as described previously^{23 (link)}. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis was performed on trypsin digests using an Ultimate 3,000 Nanosystem (Dionex, ThermoFisher Scientific) online to a Q-Exactive Quadrupole-Orbitrap instrument (Thermo Scientific)^{24 (link)}. Proteins were identified using an in‐house Mascot server (Matrix Science, London, UK). Search parameters used were as follows: enzyme; trypsin, peptide mass tolerances 10 ppm, fragment mass tolerance of 0.01 Da, 1+, 2+, and 3+ ions, with carbamidomethyl cysteine as a fixed modification and methionine oxidation as a variable modification, searching against the UniHuman Reviewed database, with a false discovery rate (FDR) of 1%, a minimum of two unique peptides per protein.

Ripmeester E.G., Caron M.M., van den Akker G.G., Surtel D.A., Cremers A., Balaskas P., Dyer P., Housmans B.A., Chabronova A., Smagul A., Fang Y., van Rhijn L.W., Peffers M.J, & Welting T.J. (2020). Impaired chondrocyte U3 snoRNA expression in osteoarthritis impacts the chondrocyte protein translation apparatus. Scientific Reports, 10, 13426.

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Quantitative Proteomics of Protein Digests

Cited 1 time

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Digests were subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Ultimate 3.000 Nanosystem (Dionex, Thermo Scientific) and Q-Exactive Quadrupole-Orbitrap instrument (Thermo Scientific) with settings as previously described (Thorpe et al., 2016 (link)). Proteins were identified using an in-house Mascot server (Matrix Science). Search parameters were as follows: enzyme; trypsin, peptide mass tolerances 10 ppm, fragment mass tolerance of 0.01 Da, 2+, and 3 + ions, with carbamidomethyl cysteine as a fixed modification and methionine oxidation as a variable modification, searching against the UniHuman Reviewed database, with a false discovery rate (FDR) of 1%, a minimum of two unique peptides per protein. Progenesis QI software (V4, Waters) was used to calculate fold changes (FC) in protein abundance. Proteins with p-value < 0.05 were considered differentially expressed (DE). All mass spectrometry raw files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD032757 and 10.6019/PXD032757 (Perez-Riverol et al., 2019 (link)).

Wijesinghe S.N., Anderson J., Brown T.J., Nanus D.E., Housmans B., Green J.A., Hackl M., Choi K.K., Arkill K.P., Welting T., James V., Jones S.W, & Peffers M.J. (2022). The role of extracellular vesicle miRNAs and tRNAs in synovial fibroblast senescence. Frontiers in Molecular Biosciences, 9, 971621.

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Quantitative Secretome Analysis by LC-MS/MS

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All media digests were randomized
and individually analyzed using liquid chromatography-tandem mass
spectrometry (LC-MS/MS) on an UltiMate 3000 Nano LC System (Dionex/Thermo
Scientific) coupled to a Q Exactive Quadrupole-Orbitrap instrument
(Thermo Scientific). Full LC-MS/MS instrument methods are described
in the Supporting Information. Tryptic
peptides, equivalent to 250 ng of protein, were loaded onto the column
and run over a 1 h gradient, interspersed with 30 min blanks (97%,
v/v) high-performance liquid chromatography grade H₂O (VWR
International), 2.9% acetonitrile (Thermo Scientific), and 0.1% TFA.
In addition to individual time points, the pooled samples for control
and treatment groups were also analyzed to investigate the differences
in the overall secretome. The mass spectrometry proteomics data have
been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD017153
and 10.6019/PXD017153.^{56 (link)} Representative
ion chromatograms are shown in Figure S4.

Anderson J.R., Phelan M.M., Foddy L., Clegg P.D, & Peffers M.J. (2020). Ex Vivo Equine Cartilage Explant Osteoarthritis Model: A Metabolomics and Proteomics Study. Journal of Proteome Research, 19(9), 3652-3667.

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Quantitative Analysis of Tau Peptides

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20 μL of sample was loaded onto a PepMap 300 C18 HPLC Column (300 um x 5 cm, 5 μm, Thermo) using an Ultimate 3000 UHPLC autosampler at a flow rate of 5 μl/min. Mobile phase consisted of 0.1% Formic Acid in water (Solvent A) and 0.1% Formic Acid in acetonitrile (Solvent B). Analyte was loaded on stationary phase for 3 minutes with 2% B, followed by a mobile phase gradient from 2–40% B over 70 minutes to elute surrogate peptides from a C18 stationary phase EASY-spray analytical column (75 um ID, 15 cm, Thermo) at a flow rate of 0.3 ul/min at 45 ⁰C. The analytical column was washed with 70% B for five minutes and re-equilibrated with 2% B for 15 minutes.
Mass spectrometry analysis was performed on a Q Exactive Quadrupole Orbitrap instrument (Thermo) operating in positive electrospray ionization mode. A parallel reaction monitoring (PRM) method with 17,500 resolution (m/z = 200), 0.7 m/z mass tolerance, automatic gain control of 2e5, and HCD fragmentation was used to analyze 5 surrogate tau peptides and their co-eluting N15-labeled internal standards with an error of < 5 ppm (S2 Table).

Self W., Awwad K., Savaryn J.P, & Schulz M. (2022). An immuno-enrichment free, validated quantification of tau protein in human CSF by LC-MS/MS. PLoS ONE, 17(6), e0269157.

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Nanoscale LC-MS/MS Peptide Analysis

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LC–MS/MS was performed using an Ultimate 3000 nano system (Dionex/Thermo Fisher Scientific) coupled online to a Q‐Exactive Quadrupole‐Orbitrap instrument (Thermo Fischer Scientific) using 10 μL aliquots of tryptic peptides equivalent to 93 ng protein per sample. Samples were randomized and run on a 1 h gradient with 30 min blanks in between runs as detailed in the Supporting Information Methods.

Kharaz Y.A., Tew S.R., Peffers M., Canty‐Laird E.G, & Comerford E. (2016). Proteomic differences between native and tissue‐engineered tendon and ligament. Proteomics, 16(10), 1547-1556.

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Label-free Quantification of Tryptic Peptides

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A NanoAcquity ultraperformance LC (Waters) coupled online to a Q-Exactive Quadrupole-Orbitrap instrument (Thermo-Fisher Scientific) was used to analyze tryptic peptides on a 2-hour gradient. 12, 28 For label-free quantification, raw files of the acquired data were analyzed (as described previously 12 ) in ProgenesisQI software (Waters), 27 (link) and the top 5 spectra for each feature were used for peptide identification in a locally implemented Mascot server (Version 2.3.01), searching against the Unihuman Reviewed database. Peptide matches above an identified threshold were adjusted to give a false discovery rate of 1% before the protein identifications were reimported into ProgenesisQI for the label-free relative quantification of unique peptides. Statistical analysis was performed using ProgenesisQI software; in brief, transformed normalized abundances were used for 1-way analysis of variance, and all peptides (with P < .05) of an identified protein were included. To select proteins for biochemical validation, the mean abundance of each protein in each of the experimental groups was calculated, and significant proteins (false discovery rate; P < .05) with a ≥±2.0-fold change between comparator groups were reported.

Hulme C.H., Peffers M.J., Harrington G.M.B., Wilson E., Perry J., Roberts S., Gallacher P., Jermin P, & Wright K.T. (2021). Identification of Candidate Synovial Fluid Biomarkers for the Prediction of Patient Outcome After Microfracture or Osteotomy. The American journal of sports medicine, 49(6).

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