The resulting peptide mixtures were analyzed in a data-dependent mode with a Q Exactive HF tandem mass spectrometer (ThermoFischer Scientific) coupled on line to an Ultimate 3000 chromatography system (ThermoFisher), essentially as previously described134 (link). A volume of 5 µL of each sample was injected, first desalted with a reverse-phase capillary precolumn C18 PepMap 100 (LC-Packing) and then resolved with a nanoscale 500 mm C18 PepMap 100 (LC-Packing) column using a 90 min gradient from 2% to 20% in 70 min and from 20% to 36% in 20 min of acetonitrile, 0.1% formic acid and then 80% for 1 min. Mass spectra of peptide ions with 2+ and 3+ charge states were acquired in the full scan range 350–1800 m/z, with a resolution set at 60,000, an AGC target at 3 × 106. MS/MS mass spectra were acquired following a Top20 method for selecting peptides for fragmentation with an AGC target set at 105, a loop count of 60 msec, an isolation window of 1.6 m/z, a resolution at 15,000, an exclusion time of 10 sec, and a scan carried out in the mass range from 200 to 2,000 m/z.
Q exactive hf tandem mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The Q-Exactive HF tandem mass spectrometer is a high-resolution instrument designed for accurate mass measurement and advanced fragmentation analysis. It utilizes Orbitrap technology to provide high-resolution, accurate mass data for qualitative and quantitative applications.
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Lab products found in correlation
Ultimate 3000 chromatography system, by Thermo Fisher Scientific (2 mentions)
Sds page loading buffer, by Beyotime (1 mentions)
Ultimate 3000 uhplc, by Thermo Fisher Scientific (1 mentions)
Easy nanolc 1000 system, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap c18 column, by Thermo Fisher Scientific (1 mentions)
Nupage 4 to 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Orbitrap analyser, by Thermo Fisher Scientific (1 mentions)
Pepmap100 c18 column, by Thermo Fisher Scientific (1 mentions)
6 protocols using q exactive hf tandem mass spectrometer
1
Tandem Mass Spectrometry for Peptide Analysis
2
Identification of PorB E3 Ubiquitin Ligases
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To identify possible E3 ubiquitin ligases involved in PorB ubiquitination, vectors expressing HA-PorB and His-Ubiquitin were co-transfected to HeLa cells and PorB was immunoprecipitated with anti-HA antibody-coated agarose beads (Lablead, #HNA-25-500). Beads were collected and heated for 10 min at 95 °C in SDS-PAGE loading buffer (Beyotime, #P0297) and subsequently send to the Beijing Genomics Institute (BGI) for further processing and identification of co-immunoprecipitated proteins by LC-MS/MS. Shortly, the sample was compressed by SDS-PAGE, dehydrated with acetonitrile, and Trypsin digested. Peptide fragments were separated on an UltiMate 3000 UHPLC (Thermo Fisher) and analyzed by a Q-Exactive HF tandem mass spectrometer (Thermo Fisher). Original MS data were loaded into Mascot 2.3.02 software and the UniProt database [https://www.uniprot.org/ ] was used for identification of human protein sequences. Percolator was used to improve and correct matching proteins. Mass spectrometry data have been deposited to the iProX repository (https://www.iprox.cn/ ) with identifier IPX0007863000 .
3
Peptide Separation and Mass Spectrometry Analysis
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The EASY-nano-LC 1000 system (Thermo Fisher Scientific, MA, USA) was used for separating peptides, which were later emitted in Q-Exactive HF tandem mass spectrometer (Thermo Fisher Scientific) by the Acclaim PepMap C18 column (3 μm, 100 Å, 75 μm × 50 cm, Thermo Fisher Scientific). Solvent A included 0.1 % formic acid supplemented within water, whereas solvent B was comprised by 0.1 % formic acid contained within 98 % acetonitrile. The injection volume was 3 μL (about 3 μg) each time, and the elution procedure (60-min) was as follows, 5-min at 5 % B, 10-min at 20 % B, 10-min at 32 % B, and 10-min at 90 % B. At last, B was reduced to 5 % within the following 60 s, followed by 10-min equilibration prior to subsequent injection.
A Thermo Q-Exactive HF mass spectrometer was programmed for obtaining 55 × 16 m/z spectra (16 m/z precursor isolation windows, resolution of 30,000, AGC target of 1e5, and maximal injection time of 55 ms) in data independent acquisition (DIA) mode for quantitative samples. We obtained precursor spectra (350–1500 m/z) at a 120,000 resolution for hitting the AGC target of 3e6. The maximal injection time was 50 ms.
A Thermo Q-Exactive HF mass spectrometer was programmed for obtaining 55 × 16 m/z spectra (16 m/z precursor isolation windows, resolution of 30,000, AGC target of 1e5, and maximal injection time of 55 ms) in data independent acquisition (DIA) mode for quantitative samples. We obtained precursor spectra (350–1500 m/z) at a 120,000 resolution for hitting the AGC target of 3e6. The maximal injection time was 50 ms.
4
Mass Spectrometry-Based Peptide Analysis
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The separated peptides were ionized by the nanoESI source and injected into a Q-Exactive HF tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA) under DDA mode. Briefly, intact peptides were detected in the Orbitrap at a resolution of 60,000 with a MS range of 350–1,500 m/z for full scan. The 20 most intense precursor ions per survey scan were selected for higher-energy collisional dissociation (HCD) fragmentation, and the resulting fragments were analyzed with the Orbitrap at a resolution of 15,000 with a fixed mass of 100 m/z. The MS was operated between MS scan and MS/MS scan with the dynamic exclusion time of 30s. The automatic gain control (AGC) was set as 3E6 for level 1 and 1E5 for level 2.
5
Proteomic Analysis of Bacterial Extracts
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Bacterial pellets were diluted in 1× LDS (Thermo) buffer at a ratio of 7.5 μL/mg. They were heated for 10 min at 99°C, sonicated for 10 min, and then subjected to bead-beating lysis as previously described (50 (link)). The extracted proteins (25 μL) were subjected to denaturing electrophoresis onto a NuPAGE 4 to 12% Bis-Tris gel (Thermo) for 4 min. For each sample, a single gel band containing the whole cellular proteome was excised and processed as described previously (51 (link)) for in-gel trypsin proteolysis. Peptides were analyzed using a Q-Exactive HF tandem mass spectrometer (Thermo) coupled to an Ultimate 3000 nanoRSLC nanoLC system (Thermo) in data-dependent acquisition mode as previously described in reference 52 (link).
6
Tandem Mass Spectrometry of Tryptic Peptides
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Tryptic peptides were subjected to tandem mass spectrometry with a Q-Exactive HF tandem mass spectrometer (Thermo Fisher Scientific, Les Ulis, France) incorporating an ultrahigh field Orbitrap analyser coupled in line to an Ultimate 3000 chromatography system (Thermo Fisher Scientific).
The tandem mass spectrometer was operated in the data-dependent acquisition mode as previously described (Klein et al., 2016) (link). Briefly, each sample (4 µL) was injected and desalted on a reversephase capillary C18 PepMap TM 100 precolumn (LC-Packing) and then peptides were resolved based on their hydrophobicity on a nanoscale 500-mm C18 PepMap 100 column (5 mm x 300 µm i.d., Thermo Fisher Scientific) using a 90-min gradient from 2.5% to 40% of 80% CH3CN, 19.9% H2O, and 0.1% formic acid. Once separated, mass spectra of the peptides were acquired in the fullscan range of 350-1800 m/z at a resolution of 60,000 and an AGC target of 3.10 6 . Peptide ions with charges of 2+ or 3+ were selected for fragmentation according to a Top20 method consisting of the sequential analysis of each of the 20 most abundant peptide ions. MS/MS mass spectra were acquired with an AGC target set at 10 4 , a loop count of 60 ms, an isolation window of 1.6 m/z, a resolution of 15,000, a dynamic exclusion of 20 s, and a scan performed over a dynamic mass range from the first mass detected up to 15 times the first mass.
The tandem mass spectrometer was operated in the data-dependent acquisition mode as previously described (Klein et al., 2016) (link). Briefly, each sample (4 µL) was injected and desalted on a reversephase capillary C18 PepMap TM 100 precolumn (LC-Packing) and then peptides were resolved based on their hydrophobicity on a nanoscale 500-mm C18 PepMap 100 column (5 mm x 300 µm i.d., Thermo Fisher Scientific) using a 90-min gradient from 2.5% to 40% of 80% CH3CN, 19.9% H2O, and 0.1% formic acid. Once separated, mass spectra of the peptides were acquired in the fullscan range of 350-1800 m/z at a resolution of 60,000 and an AGC target of 3.10 6 . Peptide ions with charges of 2+ or 3+ were selected for fragmentation according to a Top20 method consisting of the sequential analysis of each of the 20 most abundant peptide ions. MS/MS mass spectra were acquired with an AGC target set at 10 4 , a loop count of 60 ms, an isolation window of 1.6 m/z, a resolution of 15,000, a dynamic exclusion of 20 s, and a scan performed over a dynamic mass range from the first mass detected up to 15 times the first mass.
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