Q exactive hf x ms

Manufactured by Thermo Fisher Scientific

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The Q Exactive HF-X MS is a high-resolution, accurate-mass mass spectrometer designed for a wide range of analytical applications. It features a high-field Orbitrap mass analyzer, providing high-resolution, accurate-mass measurements and sensitive detection.

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9 protocols using q exactive hf x ms

UHPLC-MS/MS Analysis of Metabolites

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UHPLC-MS/MS experiments were performed by a UHPLC system (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 × 100 mm, particle size 1.7 μm, J&W Scientific, Folsom, CA, USA) coupled to Q-Exactive HFX MS (Orbitrap MS, Thermo Fisher Scientific, Waltham, MA, USA)⁴⁷. The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The flow rate was 500 μL/min. The auto-sampler temperature was 4 °C and the injection volume was 3 μL. The gradient setting: 0~0.5 min, 95% B; 0.5~7 min, 95%~65% B; 7~8 min, 65%~40% B; 8~9 min, 40% B; 9~9.1 min, 40%~95% B; 9.1~12 min, 95% B.
The QE HFX MS was used due to its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur 4.1, Thermo Fisher Scientific, Waltham, MA, USA). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The electrospray ionization (ESI) source conditions were set as following: sheath gas flow rate as 30 Arb, Aux gas flow rate as 25 Arb, capillary temperature 350 °C, full MS resolution as 60000, MS/MS resolution as 7500, collision energy as 10/30/60 in Na⁺/Ca²⁺ exchanger (NCE) mode, spray voltage as 3.6 kV (positive) or −3.2 kV (negative), respectively.

Chen J., Nie Y., Xu J., Huang S., Sheng J., Wang X, & Zhong J. (2022). Sensory and metabolite migration from tilapia skin to soup during the boiling process: fast and then slow. NPJ Science of Food, 6, 52.

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HLA-C Peptide Sequencing and Analysis

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The dataset was obtained from ProteomeXChange[36 ] (accession number: PXD006455). The experiments were conducted on two common HLA-C: HLA-C*05:01 and HLA-C*07:02. These HLA class I molecules were isolated from the cell surface of C*05 and C*07 transfected 721.221 cells, and sequenced bound peptides by mass spectrometry. As observed in the original article[18 ], HLA-C*05:01 has higher expression level and more diversified binding peptides. In our testing, we chose the binding peptides of HLA-C*05:01 (with length between 9 to 12 residues) to demonstrate the performance of our method. In total, there are 339,513 spectra acquired in a total 25 fractions of LC-MS/MS analysis using the Q Exactive HF-X MS (Thermo Fisher Scientific)[36 ].

Li S., DeCourcy A, & Tang H. (2018). Constrained De Novo Sequencing of neo-Epitope Peptides using Tandem Mass Spectrometry. Research in computational molecular biology : ... Annual International Conference, RECOMB ... : proceedings. RECOMB (Conference : 2005-), 10812, 138-153.

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Proteome and Acetylome Profiling by PRM

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Tissue collection and processing were the same as described above. For the total proteome, the tryptic peptides of each sample were separated on an EASY‐nLC 1000 UPLC system (Thermo Fisher Scientific). Subsequently, the eluted peptides were analysed using Q Exactive™ Plus MS (Thermo Fisher Scientific), with the following parameters: PRM mode, the MS scan range 406–1195 m/z, automatic gain control (AGC) target was set to 3E6 for full MS and 1E5 for MS/MS, the maximum injection time (IT) was 50 ms for full MS and 160 ms for MS/MS, and the isolation window of MS/MS was set to 1.6 m/z. For the acetylome, the enriched Kac peptides were separated on an EASY‐nLC 1200 UPLC system (Thermo Fisher Scientific). Then, the eluted peptides were analysed using Q Exactive™ HF‐X MS (Thermo Fisher Scientific) with the following parameters: PRM mode, MS scan range 430–930 m/z, AGC target was set to 3E6 for full MS and 1E5 for MS/MS, the maximum IT was 50 ms for full MS and 200 ms for MS/MS, and the isolation window of MS/MS was set to 1.4 m/z.²⁶, ²⁷ For the PRM data analysis, Skyline (v.3.6)²⁸ was used to process the obtained MS data.

Shangguan Y., Wang Y., Shi W., Guo R., Zeng Z., Hu W., Cai W., Yan Q., Xu Y., Tang D, & Dai Y. (2021). Systematic proteomics analysis of lysine acetylation reveals critical features of placental proteins in pregnant women with preeclampsia. Journal of Cellular and Molecular Medicine, 25(22), 10614-10626.

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Proteomic Analysis of Spastic CP

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The sample we tested was plasma. Four spastic CP samples and four control samples were used for proteomic analysis. A 100 μg protein sample was taken from each sample and digested overnight at 37°C. Each sample was processed and labeled with the instruction of iTRAQ Reagent-8 Multiplexing Kit (AB Sciex, UK). The labeled sample was mixed in equal volumes, desalted, and lyophilized. High-performance liquid chromatography was performed with a Rigol L3000 system and a C18 chromatographic column (Waters BEH C18, 5 mm). A Diane NCS3500 system (Thermo Science FicTM) equipped with a trap and an analytical column was used to fractionate the labeled samples, and the precursor ions decomposed by the higher-energy C-trap dissociation (HCD) method were sent to a tandem mass spectrometry Q Exactive HF-X MS (Thermo Fisher, Waltham, MA) for data acquisition and analysis.

Wang X.K., Gao C., Zhong H.Q., Kong X.Y., Qiao R., Zhang H.C., Chen B.Y., Gao Y, & Li B. (2022). TNAP—a potential cytokine in the cerebral inflammation in spastic cerebral palsy. Frontiers in Molecular Neuroscience, 15, 926791.

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High-pH Peptide Fractionation and LC-MS/MS

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Each of the 24 high-pH peptide fractions was resuspended in loading buffer (0.1% FA, 0.03% TFA, 1% ACN). Peptide eluents were separated on a self-packed C18 (1.9 μm Maisch) fused silica column [25 cm × 75 μm internal diameter (ID), New Objective] by an Easy nLC 1200 (Thermo Scientific) and monitored on an Q-Exactive HFX MS (Thermo Scientific). Elution was performed over a 120 min gradient at a rate of 300 nl/min with buffer B ranging from 3% to 40% (buffer A: 0.1% FA in water; buffer B: 0.1% FA in 80% ACN). The mass spectrometer was set to acquire data in positive ion mode using data-dependent acquisition with top 10 cycles. Each cycle consisted of one full MS scan followed by a maximum of 10 MS/MS. Full MS scans were collected at a resolution of 120,000 (400–1600 m/z range, 3 × 10̂6 AGC, 100 ms maximum ion injection time). All higher energy collision-induced dissociation (HCD) MS/MS spectra were acquired at a resolution of 45,000 (1.6 m/z isolation width, 30% collision energy, 1 × 10^–5 AGC target, 86-ms maximum ion time). Dynamic exclusion was set to exclude previously sequenced peaks for 20 s within a 10-ppm isolation window.

Wynne M.E., Lane A.R., Singleton K.S., Zlatic S.A., Gokhale A., Werner E., Duong D., Kwong J.Q., Crocker A.J, & Faundez V. (2021). Heterogeneous Expression of Nuclear Encoded Mitochondrial Genes Distinguishes Inhibitory and Excitatory Neurons. eNeuro, 8(4), ENEURO.0232-21.2021.

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TMT Quantitative Mass Spectrometry

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TMT-groups were reconstituted in MS loading buffer composed of 5% FA and 5% acetonitrile. The samples (0.75 μg) were loaded onto a Thermo Q Exactive HF-X MS using an Easy1200 nLC at a flow rate of 1 μl/min. A chip system (picoChip, New Objective) was used to trap and elute the peptides. The stationary phase was Reprosil PUR 3um 120 Å, 3 microns, 25 cm. A 60 min gradient from 5% buffer B (0.1% FA in acetonitrile) to 30% B (in 52 min) followed by a gradient from 30% B to 45% B in 8 min. Buffer A was 0.1% FA in water and buffer B was 0.1% FA in 80% acetonitrile, and the flow rate was 500 nl/min.
MS¹ settings were as follows: range 350 to 1450 m/z; resolution 120,000; Automatic Gain Control target 3e6; 50 ms maximum ion accumulation time; one scan range (single scan); and profile data were obtained. Tandem MS² scans were obtained as follows: A top 16 method was used with a fixed first mass of 100 m/z with dynamic higher mass range; isolation window was 0.8 m/z; AGC target of 1e5; 80 ms maximum ion accumulation time; collision energy of 32 normalized collision energy; and an underfill ratio of 10.0%. Dynamic exclusion was set to 15 s, and the precursor charge states 2 to 5 were permitted.

Froehlich J.W., Wang H.H., Logvinenko T., Kostel S., DiMartino S., van Bokhoven A., Moses M.A, & Lee R.S. (2021). The Urinary Proteomic Profile Implicates Key Regulators for Urologic Chronic Pelvic Pain Syndrome (UCPPS): A MAPP Research Network Study. Molecular & Cellular Proteomics : MCP, 21(1), 100176.

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Quantitative Proteomic Analysis Pipeline

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A previous method was followed (P. Liu, Guo, Mao, & Gu, 2020 (link)) with slight modifications. In brief, a Q-Exactive HF-X MS coupled with Easy-nLC 1200 (Thermo Fischer Scientific, Waltham, MA, USA) HPLC system was used for separating and detecting the peptide samples. The samples were injected into the trap column and then loaded onto a C18-reversed-phase column (75 μm × 25 cm, Thermo Fischer Scientific, USA) with a flow rate of 300 nL/min. The gradient phase contained of phase A of 0.1 % formic acid aqueous solution and phase B that was resolved using a linear gradient of 0.1 % formic acid in 84 % acetonitrile. The gradient procedure was as follows: 0–40 min, linear-gradient phase B (8–30 %); 40–50 min, B (30–100 %); 50–60 min, B (100 %).
Mass spectrometry acquisition was carried out in positive ion mode. The first-stage scanning range was set to 200–3000 m/z, achieving a resolution of 120,000 (m/z = 200). The automatic gain control (AGC) was set to 3e6; the maximum ion trapping (IT) was 200 ms. After each full scan, 20 MS2 scans were collected. The MS2 activation type was higher energy C-trap dissociation (HCD) and the isolation window was maintained at 1.5 m/z. MS2 scan resolution was fixed at 30,000 (m/z = 200) and normalized collision energy was maintained at 30 eV.

Zhang X., Zheng Y., Liu Z., Su M., Wu Z., Zhang H., Zhang C, & Xu X. (2024). Insights into characteristic metabolites and potential bioactive peptides profiles of fresh cheese fermented with three novel probiotics based metabolomics and peptidomics. Food Chemistry: X, 21, 101147.

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High-Resolution TMT Proteomics Profiling

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The TMT-10 or TMTpro-16 sample multiplex bRPLC fractions were analyzed by LC-MS/MS employing a nanoflow LC system (EASY-nLC 1200, ThermoFisher Scientific, Inc.) coupled online with a Q Exactive HF-X MS (ThermoFisher Scientific, Inc.). In brief, each sample was loaded on a nanoflow HPLC system outfitted with a reversed-phase trap column (Acclaim PepMap100 C18, 2 cm, nanoViper, ThermoFisher Scientific, Inc) and a heated (50 °C) reversed-phase analytical column (Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm × 500 mm, nanoViper, ThermoFisher Scientific, Inc). Peptides were eluted by developing a linear gradient of 2% mobile phase B (95% acetonitrile with 0.1% formic acid) to 32% mobile phase B within 120 min at a constant flow rate of 250 nL/min. High resolution (R=60,000 at m/z 200) broadband (m/z 400 – 1,600) mass spectra (MS) were acquired from which the top 12 most intense molecular ions in each MS scan were selected for high-energy collisional dissociation (HCD, normalized collision energy of 30 for TMT-10 and 34 for TMTpro) acquisition in the orbitrap at high resolution (R=45,000 at m/z 200). Charge state selection was restricted to z = +2, +3 and +4. The RF lens was set to 30% and both MS1 and MS2 spectra were collected in profile mode. Dynamic exclusion (20s) was enabled to minimize redundant selection of peptide molecular ions for HCD.

Hunt A.L., Bateman N.W., Barakat W., Makohon-Moore S., Hood B.L., Conrads K.A., Zhou M., Calvert V., Pierobon M., Loffredo J., Litzi T.J., Oliver J., Mitchell D., Gist G., Rojas C., Blanton B., Robinson E.L., Odunsi K., Sood A.K., Casablanca Y., Darcy K.M., Shriver C.D., Petricoin E.F., Rao U.N., Maxwell G.L, & Conrads T.P. (2021). Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens. iScience, 24(7), 102757.

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Peptide Analysis of Chinese White Pear

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The peptides were first analyzed by Q-exactive HF-X MS (Thermo Fisher Scientific) coupled with a Rigol L3000 HPLC system (Rigol, Inc., Beijing, China). Then, the obtained peptide sequences were searched against the reference genome of Chinese white pear (Pyrus × bretschneideri; 47,086 sequences) deposited in the NCBI database.

Wang Y., Dai M., Cai D, & Shi Z. (2020). Proteome and transcriptome profile analysis reveals regulatory and stress-responsive networks in the russet fruit skin of sand pear. Horticulture Research, 7, 16.

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