Q exactive plus orbitrap mass spectrometer

Manufactured by Thermo Fisher Scientific

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The Q Exactive Plus Orbitrap mass spectrometer is a high-resolution, accurate-mass instrument designed for a wide range of analytical applications. It combines quadrupole precursor ion selection with high-resolution, accurate-mass Orbitrap detection for precise mass measurements and structural information.

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155 protocols using q exactive plus orbitrap mass spectrometer

Proteomic Analysis of Leishmania donovani

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Proteins were extracted from the same batch of L. donovani samples used for the genomic DNA isolation. In brief, the purified promastigotes were incubated in lysis buffer (8 M urea and 1% protease inhibitor cocktail) at 4 °C for 3 min, sonicated on ice for three times using a high intensity ultrasonic processor (Scientz, Ningbo, China). Sonicated samples were centrifuged at 12,000× g at 4°C for 10 min to remove debris. Supernatant was collected, and the protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific).
For each of the L. donovani samples (HCZ isolate, DD8 and 9044 strains), proteins were subjected to an in-solution reduction, alkylation and digestion approach. In brief, 300 µg protein aliquots (n = 3) was reduced with 5 mM dithiothreitol at 56 °C for 30 min, alkylated with 11 mM iodoacetamide at room temperature in darkness for 15 min, digested with trypsin (1:50 trypsin-to-protein) at 37 °C overnight, and further digested with trypsin (1:100 trypsin-to-protein) at 37 °C for 4 h. The processed samples were dissolved in 1.0% (v/v) formic acid, and then subjected to liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) analysis using a Q ExactiveTM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled online to the EASY-nLC 1000 UPLC system.

Zheng Z., Chen J., Ma G., Satoskar A.R, & Li J. (2020). Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance. Parasites & Vectors, 13, 510.

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Proteomic Analysis of Leishmania Strains

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The protein data of L. donovani 9044 and DD8 strains was generated by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) in our previous studies (26 (link)). Proteomic data was not employed for immunoinformatics analysis in previous studies and it can be retrieved from ProteomeXchange Consortium via the accession number PXD017089. In brief, L. 9044 and L. DD8 were cultured and collected to incubate in lysis buffer (8 M urea and 1% protease inhibitor cocktail) at 4°C for 3 min. Lysis samples were sonicated on ice three times using a high-intensity ultrasonic processor (Scientz, Ningbo, China) and the supernatant was collected by centrifugation for an insolution reduction, alkylation, and digestion approach. The processed samples were dissolved in 1.0% (v/v) formic acid, and then subjected to liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis using a QExactiveTM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled online to the EASY-nLC 1000 UPLC system. Perseus software v.1.6.15.0 was employed to determine differentially expressed proteins (Fold change ≥ 3, q-value < 0.01) between L. 9044 and L. DD8.

Zhang J., Li J., Hu K., Zhou Q., Chen X., He J., Yin S., Chi Y., Liao X., Xiao Y., Qin H., Zheng Z, & Chen J. (2022). Screening Novel Vaccine Candidates for Leishmania Donovani by Combining Differential Proteomics and Immunoinformatics Analysis. Frontiers in Immunology, 13, 902066.

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Synthesis of BTZ043 and Derivatives

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Starting materials were purchased and used as received. Solvents were distilled prior to use and stored over 4 Å molecular sieves. Column chromatography was carried out using Merck silica gel 60 (63–200 µm). Flash chromatography was performed on a puriFlash^® 430 instrument (Interchim, Montluçon, France). Prepacked columns with silica gel (30 μm) were used. The maximum compound load per column was 5% (m/m) of the silica gel quantity. The synthesis of BTZ043 is described elsewhere [27 ]. Melting points (uncorrected) were determined on a Boëtius hot-stage apparatus (VEB Kombinat, NAGEMA, Dresden, GDR). NMR spectra were recorded on an Agilent Technologies VNMRS 400 MHz and a Varian INOVA 500 NMR spectrometer. Chemical shifts are reported relative to the residual solvent signal (chloroform-d: δ_H = 7.26 ppm, δ_C = 77.16 ppm; methanol-d₄: δ_H = 3.31 ppm, δ_C = 49.00 ppm; DMSO-d6 δ_H = 2.50 ppm, δ_C = 39.52 ppm). Abbreviations: s = singlet, bs = broad singlet, d = doublet, dd = doublet of doublets, m = multiplet. ESI high-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer for 2b, 2c and 2e, and on a Thermo Scientific Q Exactive^TM Plus Orbitrap mass spectrometer for 2a and 2d.

Madikizela B., Eckhardt T., Goddard R., Richter A., Lins A., Lehmann C., Imming P, & Seidel R.W. (2021). Synthesis, structural characterization and antimycobacterial evaluation of several halogenated non-nitro benzothiazinones. Medicinal Chemistry Research, 30(8), 1523-1533.

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Proteomic Analysis of Frozen Leaf Samples

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Frozen leaf samples (approximately 1.0 g for each replicate) were ground to a fine powder using liquid nitrogen, and were then lysed ultrasonically. An equal volume of Tris-phenol was added, and the samples were centrifuged at 12,000× g for 10 min at 4 °C. Proteins were precipitated, washed with methanol and acetone, and the final pellet was resuspended in 1% SDS. Protein concentrations were determined and then digested according to a previously described procedure [18 (link)]. LC-MS was performed using the Q Exactive TM Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) and the Easy-nLCTM 1200 system (Thermo Fisher Scientific). Parameters were set as follows: electrospray voltage, 2.1 kV; automatic gain control (AGC), 5E4; survey scans were acquired at a resolution of 1,200,000; resolution for HCD spectra, 15,000.

Ding K., Jia Z., Rui P., Fang X., Zheng H., Chen J., Yan F, & Wu G. (2023). Proteomics Identified UDP-Glycosyltransferase Family Members as Pro-Viral Factors for Turnip Mosaic Virus Infection in Nicotiana benthamiana. Viruses, 15(6), 1401.

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Reverse-phase LC-MS/MS Peptide Analysis

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Desalted peptides were loaded on a reverse-phase C₁₈ column (75 μm × 20 cm, 1.9 μm, 120 Å) using an EASY-nLC 1200 system (Thermo Fisher Scientific Inc.). The LC-MS/MS run time was set to 60 min with flow rate of 300 nL/min. Mobile phases A (water:0.1% HCOOH, V/V) and B (ACN:0.1% HCOOH, V/V) were used for gradient elution: 5%–25% B for 28 min, 25%–40% B for 15 min, and 40%–100% B for 5 min. Samples were analyzed on Q Exactive^TM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.). The mass spectrometer was operated in the positive ion mode and the spectra were acquired in the data-dependent acquisition mode. Full MS scans were acquired with 70,000 resolution and a scan mass range of m/z 355−2000. An Automatic Gain Control (AGC) target was set to 3e⁶ with a maximum injection time of 20 ms. A data-dependent MS/MS (dd-MS/MS) scan was acquired with 17,500 resolution. The AGC target was set to 5e⁴ with the maximum injection time defined as 50 ms. The data-dependent method was set to isolation and fragmentation of the 20 most intense peaks defined in the full MS scan. Parameters for isolation or fragmentation of selected ion peaks were set as follows: isolation width, 1.8 Th and higher energy collisional dissociation (HCD) normalized collision energy, 27%.

Zhang Y., Cai Q., Luo Y., Zhang Y, & Li H. (2022). Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity. Journal of Pharmaceutical Analysis, 13(1), 63-72.

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Peptide Analysis by LC-MS/MS

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To dissolve the lyophilized peptide samples, 0.1% formic acid (solvent A) was used, and the samples were then directly loaded onto a reversed-phase analytical column (15 cm in length and 75 μm inside diameter (i.d.)). The peptide mixture was separated using a linear gradient of solvent B (0.1% formic acid in 98% acetonitrile) at a constant flow rate of 400 nL/min. In this study, we performed LC-MS using the Q Exactive ^TM Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) and the Proxeon Biosystems Easy nLC^TM system (Thermo Fisher Scientific). Parameters were set as follows: electrospray voltage, 2.0 kV; automatic gain control (AGC), 5E4; survey scans were acquired at a resolution of 70,000; resolution for HCD spectra, 17,500; isolation width, 2 m/z.

Hu H., Cai L., Zhang T., Liu T., Jiang Y., Liu H., Lu Q., Yang J, & Chen J. (2022). Central Role of Ubiquitination in Wheat Response to CWMV Infection. Viruses, 14(8), 1789.

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Targeted Metabolomics Analysis by HILIC-MS

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Analytical separation and quantification were performed on a Dionex^TM UltiMate^TM 3000 system (Thermo Fisher Scientific, Dreieich, Germany) equipped with a SeQuant ZIC-HILIC column (5 μm, 2.1 × 150 mm, SeQuant with guard column, Merck, Darmstadt, Germany). Mass spectra were recorded on a Q-Exactive^TM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). The electrospray ionization conditions are given in [7 (link)]. The column temperature was set to 25 °C. Mass measurements were performed in the HESI-positive mode, full scan mode from 75 to 200 m/z, at a resolution of 70,000. For qualitative MS/MS analysis, the collision energy was set to 35 V and data were collected in DIA (Data Independent Acquisition) mode.

Fenizia S., Weissflog J, & Pohnert G. (2021). Cysteinolic Acid Is a Widely Distributed Compatible Solute of Marine Microalgae. Marine Drugs, 19(12), 683.

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Arabidopsis Phosphoproteome Profiling

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Phosphopeptide samples were separately analyzed by a QExactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) LC-MS/MS for a total of 18 runs at the UC Davis Proteomics Core. LC-MS/MS settings and specifics are described in Methods S1. LC-MS/MS raw files were analyzed using the Maxquant (version 1.5.1.0) and Perseus (version 1.5.0.15) software packages (Tyanova et al., 2016a (link); Tyanova et al., 2016b (link)). Raw data files were searched against a FASTA database containing the Arabidopsis thaliana proteome. Statistical analysis is described in Methods S1. Raw data and search engine results are available at the PRIDE ProteomeXchange website under accession number PXD010440 (Vizcaino et al., 2013 (link)). Details of quantified phosphosites are shown in Tables S1–S4. Search parameters for Maxquant can be found in Table S5. Highest intensities and spectra per phosphosite are reported in Table S6.

Kadota Y., Liebrand T.W., Goto Y., Sklenar J., Derbyshire P., Menke F.L., Torres M.A., Molina A., Zipfel C., Coaker G, & Shirasu K. (2018). Quantitative phosphoproteomic analysis reveals common regulatory mechanisms between effector- and PAMP-triggered immunity in plants. The New phytologist, 221(4), 2160-2175.

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Quantification of Bioactive Compounds in Schisandra acutum

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The BIAs in S. acutum were identified and quantified using the Waters ACQUITY Ultra-Performance Liquid Chromatography (UPLC) System (Waters, Milford, MA, United States) and the Q Exactive Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific., Waltham, MA, United States). Dried powder samples (0.25 g) of the root, leaf, stem, and seed of S. acutum were accurately weighted and subsequently extracted with 20 mL of methanol solution for 30 min in an ultrasonic bath. Chromatographic separation was carried out on the Waters ACQUITY UPLC system equipped with a BEH C18 column (2.1 × 100 mm, 1.7 µm), and the column temperature was kept at 30°C. The mobile phases were water with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The solvent gradients for B were 0–6 min, 5%; 6–20 min, 5–16%; 20–22 min, 16–95%; 22 –25 min, 95%; and 25–26 min, 5%. The Q Exactive MS system used the electrospray ionization source (ESI) in the positive ion mode. The spray voltage and capillary temperature were 3.0 kV and 320 °C, respectively. The flow rates of the atomization gas and heating auxiliary gas were set at 35 arb and eight arb, respectively. The auxiliary gas heating temperature was set at 350°C.

Yang Y., Sun Y., Wang Z., Yin M., Sun R., Xue L., Huang X., Wang C, & Yan X. (2022). Full-length transcriptome and metabolite analysis reveal reticuline epimerase-independent pathways for benzylisoquinoline alkaloids biosynthesis in Sinomenium acutum. Frontiers in Plant Science, 13, 1086335.

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Automated Phosphopeptide Enrichment

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Phosphopeptide enrichment was carried out on the HILIC fractions with titanium IMAC (immobilised metal affinity chromatography) beads (MagReSyn) using the original protocol by Larsen et al.^{43 (link)} on an automated bead processing robot (KingFisher, Thermo Fisher Scientific) as described in Tape et al.^{55 (link)}. The enriched phosphopeptide fractions were dried, redissolved in 5% formic acid and analysed in data-dependent mode on Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific).

Offenburger S.L., Bensaddek D., Murillo A.B., Lamond A.I, & Gartner A. (2017). Comparative genetic, proteomic and phosphoproteomic analysis of C. elegans embryos with a focus on ham-1/STOX and pig-1/MELK in dopaminergic neuron development. Scientific Reports, 7, 4314.

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