Q exactive plus mass spectrometer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Canada, Australia, Japan

The Q Exactive Plus mass spectrometer is a high-resolution, accurate-mass (HRAM) instrument designed for qualitative and quantitative analysis. It features Orbitrap mass analyzer technology, providing high-resolution, high-mass accuracy, and high-sensitivity performance.

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467 protocols using q exactive plus mass spectrometer

Peptide Fractionation and Mass Spectrometry

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HPLC fractionation was conducted an Agilent 300Extend-C18 column (4.6 × 250 mm, 5 μm; Agilent Technologies, USA) as described by Zhan et al. [20 (link)]. Briefly, the peptides were first separated with a gradient of 8 to 32% acetonitrile (pH 9.0) at a flow rate of 0.5 mL min^− 1 over 60 min into 60 fractions. Then, the collected 60 fractions were pooled into 18 ponds and dried by vacuum centrifugation.
LC − MS/MS was carried out with an automated Easy-nLC 1000 UPLC system coupled to a Q-Exactive™ Plus mass spectrometer (Thermo Fisher Scientific, USA) as described previously [30 , 31 ]. In brief, the resulting peptides were dissolved in 0.1% formic acid (solvent A) and eluted using solvent B (0.1% formic acid in 98% acetonitrile) in a linear gradient comprised of an increase from 6 to 23% over 26 min, 23 to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min on an Easy-nLC 1000 UPLC system. And then, the peptide samples were analyzed on a Q-Exactive™ Plus mass spectrometer (Thermo Fisher Scientific, USA) equipped with the NanoSpray Ionization (NSI) source coupled with a UPLC system. The working parameters of the mass spectrometer were selected according to previous studies [31 , 68 (link)].

Wang Z.Q., Zhao Q.M., Zhong X., Xiao L., Ma L.X., Wu C.F., Zhang Z., Zhang L.Q., Tian Y, & Fan W. (2020). Comparative analysis of maca (Lepidium meyenii) proteome profiles reveals insights into response mechanisms of herbal plants to high-temperature stress. BMC Plant Biology, 20, 431.

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Time-Resolved Metabolomic Profiling Workflow

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For metabolomic profiling, roughly 7.5 x 10⁷ cells were collected at each time-point (0, 0.5, 1, 2.5, 5, 7.5, 10, 15, and 30 minutes), filtered onto a nylon membrane, and quenched in an 80:20 mixture of HPLC-grade methanol and HPLC-grade water at -20°C. This was allowed to chill at -20°C for at least 20 minutes before the cell material slurry was repeatedly pipetted over the filter and collected into a microcentrifuge tube. The extraction solvent/cell slurry was centrifuged at 4°C for 5 minutes, and a fraction of this supernatant was dried under nitrogen gas for resuspension and metabolite profiling using liquid chromatography coupled to mass spectrometry (LC-MS). Each sample was examined using two different analytical separation methods. One method included reverse-phase ion-pairing LC on an Agilent Extend C18 column with tributylamine as an ion-pairing agent, followed by metabolite identification using a Q Exactive Plus mass spectrometer (Thermo Scientific). The other method included LC on a ZIC pHILIC column (SeQuant) followed by metabolite identification using a Q Exactive Plus mass spectrometer (Thermo Scientific)[72 (link)–74 (link)]. Data were then analyzed using the open-source software Metabolomic Analysis and Visualization ENgine (MAVEN)[75 (link)].

Gibney P.A., Chen A., Schieler A., Chen J.C., Xu Y., Hendrickson D.G., McIsaac R.S., Rabinowitz J.D, & Botstein D. (2020). A tps1Δ persister-like state in Saccharomyces cerevisiae is regulated by MKT1. PLoS ONE, 15(5), e0233779.

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LC-PRM Analysis of Desalted Peptides

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After the enzymatically hydrolyzed peptides were desalted by the Strata X C18 desalting column, a hybrid Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled to a nano-EASY-nLC 1000 UPLC system (Thermo Fisher Scientific) was used to perform LC-PRM analysis. Solvent B containing 0.1 % formic acid in 80 % acetonitrile had an increasing gradient from 2 % to 40 %, and then to 90 %, on an EASY-nLC 1000 UPLC system with 300 nL/min CFR. After chromatographic separation, the sample was analyzed by a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The scanning range of the precursor ion was 350–1500 m/z, the resolution of the primary mass spectrum was 70,000, and the ion dynamic exclusion time was 30 s. Normalized collision energy was set to 30 eV for intact fraction detection.

Shang Q.X., Yang Y.S., Zhang H.L., Cheng Y.P., Lu H., Yuan Y., Chen L.Q, & Ji A.F. (2023). Vitamin D receptor induces oxidative stress to promote esophageal squamous cell carcinoma proliferation via the p53 signaling pathway. Heliyon, 10(1), e23832.

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Shotgun Proteomics Workflow for Mass Spectrometry

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Cells were lysed in 6 M urea and 2 M thiourea in 100 mM Tris-HCl (pH 8.5). Ten ug of protein were reduced with 1 mM DTT for 30 min and alkylated with 5 mM iodoacetamide for 30 min in the dark. The lysates were diluted 4-fold with 50 mM ammonium bicarbonate, followed by overnight digestion with sequencing grade trypsin. Resulting peptides were acidified with trifluoroacetic acid. Peptides were resuspended in 2% acetonitrile/0.1% TFA prior to the LC-MS/MS analysis. Peptides were analyzed by liquid-chromatography using the EASY-nLC1000 UHPLC (Thermo Fisher Scientific) coupled with the Q-Exactive (QE)-Plus mass spectrometer. Peptides were separated on 75 μm i.d. × 50 cm long EASY-spray PepMap columns packed with 2 μm C18 beads with 100Å pore size. MS acquisition was performed in a data-dependent manner using the positive-ion mode—with a selection of the top ten peptides—and MS/MS analysis. Full MS spectra were acquired at a resolution of 70,000, m/z range of 300–1800 Th, with an AGC target of 3 × 10⁶ ions and maximal injection time of 20 milliseconds (ms). MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 1 × 10⁵. Dynamic exclusion was set to 30 s.

Nagaraj K., Sarfstein R., Laron Z, & Werner H. (2022). Long-Term IGF1 Stimulation Leads to Cellular Senescence via Functional Interaction with the Thioredoxin-Interacting Protein, TXNIP. Cells, 11(20), 3260.

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Mass Spectrometry of Denatured and Native Antibodies

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For denatured antibody analysis, samples were analyzed by a Q Exactive (QE) Plus mass spectrometer (Thermo Fisher Scientific) coupled to an Ion Max source (Thermo Fisher Scientific) fitted with a HESI II probe with a flow rate of 25–200 μL/min. Mass spectra were acquired at a resolution setting of 17,500 with a scan range from m/z 1,600 to 6,000 and settings including a spray voltage of 3.7 kV, a sheath-gas flow rate of 7, an in-source collision-induced-dissociation (SID) value of 20 eV, and a capillary temperature of 320 °C. For native antibody analysis, samples were analyzed by a QE UHMR mass spectrometer (Thermo Fisher Scientific) coupled to an Ion Max source fitted with a HESI II probe with a flow rate of 100 μL/min. Mass spectra were acquired at a resolution setting of 12,500 with a scan range from m/z 2,500 to 15,000 and settings including a spray voltage of 4.3 kV, a sheath-gas flow rate of 20, and a capillary temperature of 275 °C. Ion transfer optics throughout the instrument and in-source trapping fragmentation voltage were tuned for every sample specifically. For the noncovalent antibody complex, 125 °C of probe heater temperature was applied. Mass spectra were viewed in Thermo Xcalibur Qual Browser software (Thermo Fisher Scientific).

Park H.M., Winton V.J., Drader J.J., Manalili Wheeler S., Lazar G.A., Kelleher N.L., Liu Y., Tran J.C, & Compton P.D. (2020). Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry. Analytical chemistry, 92(2), 2186-2193.

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Coculture Phosphoproteomics of Myeloma Cells

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For coculture experiments, 5×10⁶ HS5 cells were seeded into a T75 flask. Seventeen hours later, cultures were washed with phosphate buffered saline (PBS), before the addition of 10⁷ MM.1S mC/Luc cells. Twenty-four hours later, cocultures were treated with 1 μM tofacitinib for 1.5 hours and 24 hours. MM.1S cells in suspension were harvested by aspiration, centrifuged, washed with PBS, and flash-frozen prior to analysis. For untreated MM.1S monoculture or HS5 monoculture experiments, 10⁷ cells were used. For sample preparation, frozen cell pellets were lysed in 8 M urea. 1 mg of total protein was then reduced in tris(2-car-boxyethyl)phosphine (TCEP) and free cysteines alkylated with iodoacetamide. Proteins were then digested at room temperature for 18 hours with trypsin. Peptides were desalted, lyophilized, and enriched for phosphopeptides using immobilized-metal affinity column (IMAC) with Fe-NTA loaded beads.^{25 (link)} Phosphopeptides were analyzed on a Thermo Q-Exactive Plus mass spectrometer coupled to a Dionex Ultimate 3000 NanoRSLC liquid chromatography instrument with 3.5 hour linear gradient. Raw proteomic data files are available at the ProteomXchange PRIDE repository (Accession number PXD006581). Additional details are provided in the Online Supplementary Methods.

Lam C., Ferguson I.D., Mariano M.C., Lin Y.H., Murnane M., Liu H., Smith G.A., Wong S.W., Taunton J., Liu J.O., Mitsiades C.S., Hann B.C., Aftab B.T, & Wiita A.P. (2018). Repurposing tofacitinib as an anti-myeloma therapeutic to reverse growth-promoting effects of the bone marrow microenvironment. Haematologica, 103(7), 1218-1228.

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Metabolite Identification using Mass Spectrometry

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Feature annotation was performed considering a ± 10 ppm mass tolerance and using our in-house spectral database [26 (link), 28 (link)], as well as the publicly available databases KEGG [29 (link)], HMDB [30 (link)] and METLIN [31 (link)]. To be identified, ions had to match at least two orthogonal criteria among accurate measured mass, isotopic pattern, MS/MS spectrum, and retention time; and to those of an authentic chemical standard analyzed under the same analytical conditions, as proposed by the Metabolomics Standards Initiative [32 (link)]: level 1 (identified): based on accurate mass, column retention time similarity with a standard and MS/MS spectrum. Level 3 (putatively characterized): based on accurate mass, and interpretation of MS/MS spectra. Metabolite identification was further confirmed by additional LC/ESI–MS–MS experiments, performed using a Dionex Ultimate chromatographic system combined with a Q-Exactive Plus mass spectrometer (Hilic) or a Fusion mass spectrometer (C18), under nonresonant collision-induced dissociation conditions using higher-energy C-trap dissociation (HCD), at normalized collision energies (NCEs) 10, 20, 40 and 80%.

Adel-Patient K., Lezmi G., Castelli F.A., Blanc S., Bernard H., Soulaines P., Dumond P., Ah-Leung S., Lageix F., de Boissieu D., Cortes-Perez N., Hazebrouck S., Fenaille F., Junot C, & Dupont C. (2018). Deep analysis of immune response and metabolic signature in children with food protein induced enterocolitis to cow’s milk. Clinical and Translational Allergy, 8, 38.

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Proteomics Analysis of OmRV Protein Bands

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The four major SDS-PAGE-separated protein bands from OmRV fraction 12 were analysed by MS/MS for peptide identification. The proteins were reduced, alkylated and in-gel digested with trypsin (Roche Applied Science) overnight as described elsewhere (http://www.scilifelab.se/facilities/bioanalytical-proteomics/). The digested products were extracted by sonication in 5% formic acid and 60% acetonitrile. The peptides were separated by reversed-phase chromatography on a C18-column in a 15 mL 0.1% formic acid acetonitrile gradient (A: 0% and B: 99.9% acetonitrile), electrosprayed on-line to a Q Exactive Plus mass spectrometer (Thermo Finnigan) and sequentially analysed by MS/MS tandem spectrometry. Obtained data were analysed by Proteome Discover 1.4 (Thermo Fisher Scientific) with an in-house FASTA database containing proteins from OmRV, and other possible contaminations such as serum Bos Taurus, Escherichia coli and Homo sapiens downloaded from UniprotKB. The search criteria for protein identification were set to at least two matching peptides at a 95% confidence level per protein. Proteins were identified, and scores calculated, by SEQUEST52 (link).

Okamoto K., Miyazaki N., Larsson D.S., Kobayashi D., Svenda M., Mühlig K., Maia F.R., Gunn L.H., Isawa H., Kobayashi M., Sawabe K., Murata K, & Hajdu J. (2016). The infectious particle of insect-borne totivirus-like Omono River virus has raised ridges and lacks fibre complexes. Scientific Reports, 6, 33170.

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Proteomic Analysis of EVs and MPs

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Analysis of EVs and MPs was performed as described previously (18 (link)). Briefly, a urea-containing buffer was used to lyse EVs and MPs. Proteins were reduced, alkylated, and digested in solution by trypsin. A Pierce C18 Spin Column (Thermo Fisher Scientific) was used to purify the sample that was dried and resolved in 0.1% formic acid. Peptides were separated in reversed phase on a C18-column and electrosprayed on-line to a Q Exactive Plus mass spectrometer (Thermo Finnigan). Tandem mass spectrometry (MS/MS) was performed applying higher-energy collisional dissociation (HCD). The Sequest algorithm in Proteome Discoverer 1.4 (Thermo Fisher Scientific) was used to search databases toward a FASTA database of TIGR4 (for serotype 4) or SP3-BS71 (for serotype 3) proteins from UniProtKB. Criteria for protein identification were at least two matching peptides of 95% confidence level. In order to avoid false-positives, only proteins with a score of 20 or above were included in the analysis. Subcellular localizations of proteins were predicted as before (18 (link)).
For analysis of the IP samples, the following mass spectrometry setup was used.

Narciso A.R., Iovino F., Thorsdottir S., Mellroth P., Codemo M., Spoerry C., Righetti F., Muschiol S., Normark S., Nannapaneni P, & Henriques-Normark B. (2022). Membrane particles evoke a serotype-independent cross-protection against pneumococcal infection that is dependent on the conserved lipoproteins MalX and PrsA. Proceedings of the National Academy of Sciences of the United States of America, 119(23), e2122386119.

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Proteomics Sample Preparation and Analysis

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NpFdx, NpDps, and EcDps proteins were reduced, alkylated and in-gel digested by trypsin according to a standard operating procedure [55] . Thereafter the samples were dried and dissolved in 15 µL of 0.1% formic acid. The peptides were separated in reversed-phase on a C18-column and electrosprayed on-line to Q Exactive Plus mass spectrometer (Thermo Finnigan). Tandem mass spectrometry was performed applying higher-energy C-trap dissociation. Omega [28, 53] .

Moparthi V.K., Moparthi S.B., Howe C., Raleiras P., Wenger J, & Stensjö K. (2019). Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA. Biochimica et biophysica acta. Bioenergetics, 1860(10).

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