Q exactive hf instrument

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The Q Exactive HF is a high-resolution mass spectrometer designed for accurate and sensitive analysis of a wide range of samples. It features a quadrupole-Orbitrap hybrid architecture, enabling both precursor ion selection and high-resolution, accurate-mass (HRAM) detection of analytes.

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17 protocols using q exactive hf instrument

Nanoflow LC-MS/MS for Protein Quantification

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All MS analyses were conducted on a Q Exactive HF instrument (Thermo Fisher Scientific) in positive mode. Chromatography was accomplished using a nanoAcquity UPLC, equipped with a nanoAcquity NPLC Symmetry C18 trap column, and an Acquity UPLC Peptide BEH C18 analytical column (Waters), heated to 37 °C, and a Triversa Nanomate source (Advion). We used 1% acetonitrile/0.1% formic acid for mobile phase A and 99% acetonitrile/0.1% formic acid for mobile phase B. We trapped analytes at 4 μl/min for 4 min in mobile phase A. Separation was done at 0.5 μl/min flow rate using the following gradient: 2 to 5% mobile phase B over 0 to 3 min, 5 to 40% B over 3 to 93 min, 40% B over 93 to 98 min, 40 to 98% B over 98 to 100 min, 98% B over 100 to 105 min, 98 to 2% B over 105 to 106 min, and 2% B over 106 to 120 min.

Chang D., Klein J., Hackett W.E., Nalehua M.R., Wan X.F, & Zaia J. (2022). Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry. Molecular & Cellular Proteomics : MCP, 21(11), 100412.

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Peptide Separation and Mass Spectrometry

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Peptide lysates were separated using a 120 min nonlinear gradient from 3.2 to 40% (v/v) acetonitrile, 0.1% (v/v) formic acid on an analytical column (Acclaim PepMap100, 75 μm inner diameter, 25 cm, C18, Thermo Scientific) in a UHPLC system (Ultimate 3000 RSLCnano; Thermo Fisher Scientific, Idstein, Germany). Mass spectrometry was performed on a Q Exactive HF instrument (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a TriVersa NanoMate (Advion, Ltd., Harlow, UK) source in LC chip coupling mode. The following MS settings were selected: loop count 10, normalized higher‐energy collisional dissociation (HCD) 28%, MS scans in the Orbitrap (resolution 120 000, scan range 350–1550 m/z, ion count target 3 × 10⁶, injection time 100 ms), MS/MS scans in the quadrupole (isolation window of 1.4 m/z, resolution 15 000, scan range 200–2000 m/z) and dynamic exclusion 30 s.

Lechner U., Türkowsky D., Dinh T.T., Al‐Fathi H., Schwoch S., Franke S., Gerlach M., Koch M., von Bergen M., Jehmlich N, & Dang T.C. (2018). Desulfitobacterium contributes to the microbial transformation of 2,4,5‐T by methanogenic enrichment cultures from a Vietnamese active landfill. Microbial Biotechnology, 11(6), 1137-1156.

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Protein Digestion and Mass Spectrometry

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The protein samples eluted from Strep-Tactin affinity resin were subjected to in-solution digestion. The pH value of the protein and control samples was adjusted to 8.0 by adding 500 mM Tris-HCl (pH 8.0), which was followed by reduction in 10 mM DTT for 30 min and alkylation in 30 mM iodoacetamide for 30 min in the dark. Five-fold volumes of chilled acetone were added into the samples for protein precipitation. The protein pellets were resuspended in 20 μL of 25 mM ammonium bicarbonate and then digested overnight with 1:50 (w/w) MS-grade trypsin at 37 °C. After desalting with self-parked StageTips, the peptide samples were separated on a self-packed column (75 μm id, 15 cm, C18-AQ, 1.9 μm, Dr Maisch) by an EASY-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, USA) at a flow rate of 250 nL/min. The eluates were directly analyzed by a Q-Exactive HF instrument (Thermo Fisher Scientific, Waltham, MA, USA) using a data-dependent acquisition mode. The raw data were searched against the database using Proteome Discoverer 2.2 (Thermo Fisher Scientific, Waltham, MA, USA). The decoy database searches were also performed in parallel, and peptides and proteins less than 1% false discovery rate (FDR) were accepted.

Tang M., Xia Y., Xiao T., Cao R., Cao Y, & Ouyang B. (2022). Structural Exploration on Palmitoyltransferase DHHC3 from Homo sapiens. Polymers, 14(15), 3013.

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SUMO Modification Site Identification

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Identification of SUMO modification lysine sites was carried out as described before (42 , 43 , 44 (link)). Briefly, HEK293 cells were transfected with mSUMO3 and HA-GβL, followed by denaturating Ni–NTA pulldown as described previously. The Ni–NTA resin was extensively washed with 50 mM ammonium bicarbonate to remove traces of Triton, and the proteins were digested with trypsin directly on the Ni–NTA solid support for 16 h at 37 °C. The mSUMO3-modified peptides were immunoprecipitated with a custom anti-NQTGG antibody that recognizes the tryptic remnant created on the SUMO-modified lysine side chain, as described before (42 , 43 , 44 (link)). Samples were analyzed on the Q-Exactive HF instrument (ThermoFisher Scientific), and raw files were processed using MaxQuant and Perseus, as described previously (42 , 43 , 44 (link)).

Park S.L., Ramírez-Jarquín U.N., Shahani N., Rivera O., Sharma M., Joshi P.S., Hansalia A., Dagar S., McManus F.P., Thibault P, & Subramaniam S. (2024). SUMO modifies GβL and mediates mTOR signaling. The Journal of Biological Chemistry, 300(4), 105778.

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Nano-HPLC-MS/MS Proteomics Workflow

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Online chromatography was performed with a Thermo EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) coupled online to the Q Exactive HF instrument with a nano-electrospray ion source (Thermo Fisher Scientific). For each samples, 1 μg of peptides was injected onto a 50 cm column (EASY-Spray column, 50 cm × 75 µm ID, PepMap C18, 2 µm particles, 100 A pore size - ES803 - Thermo Fisher Scientific) and separated with a multi-step gradient from 2 to 23% acetonitrile in 135 min and 23 to 45% acetonitrile in 20 min, at a flow rate of 250 nL/min over 190 min. Column temperature was set to 50 °C. MS data were acquired using Xcalibur software, using a data-dependent top10 method with a survey scans (300–1700 m/z) at a resolution of 60,000, and a MS/MS scans (fixed first mass 100 m/z) at a resolution of 15,000. The AGC target and maximum injection time for the survey scans and the MS/MS scans were set to 3 E⁶, 100 ms and 1 E⁵, 45 ms, respectively. The isolation window was set to 1.6 m/z and normalized collision energy fixed to 28 for HCD fragmentation. We used an underfill ratio of 2.0% for an intensity threshold of 4.4 E⁴. Unassigned precursor ion charge states as well as 1, 8 and >8 charged states were rejected and peptide match was disable. Exclude isotopes was enabled and selected ions were dynamically excluded for 45 seconds.

Lago M., Monteil V., Douche T., Guglielmini J., Criscuolo A., Maufrais C., Matondo M, & Norel F. (2017). Proteome remodelling by the stress sigma factor RpoS/σS in Salmonella: identification of small proteins and evidence for post-transcriptional regulation. Scientific Reports, 7, 2127.

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Proteomic Analysis of Phosphorylated Peptides

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Tryptic peptides from in-gel digestion were analyzed on a Q Exactive plus instrument (Thermo Fisher Scientific) and phosphoenrichment peptides on a Q Exactive HF instrument (Thermo Fisher Scientific), both instruments coupled with an EASY nLC 1200 chromatography system (Thermo Fisher Scientific). Sample was loaded on an in-house packed 25-cm (for in-gel digestion) and 53-cm (phosphoenrichment) nano-HPLC column with C₁₈ resin (1.9-μm particles, 100-Å pore size; Reprosil-Pur Basic C18-HD resin; Maisch GmbH, Ammerbuch-Entringen, Germany) after an equilibration step in 100% solvent A (H₂O, 0.1% FA). Peptides were first eluted using a 2% to 5% gradient of solvent B (ACN, 0.1% FA) during 5 min, a 5% to 10% gradient during 20 min, a 10% to 30% gradient during 70 min, and finally a 30% to 60% gradient during 20 min, all at 300 nl·min⁻¹ flow rates. The instrument method was set up in the data-dependent acquisition mode. After a survey scan in the Orbitrap (resolution, 70,000 and 60,000), the 10 most intense precursor ions were selected for HCD fragmentation with normalized collision energy set to 27 and 28. Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1, 7, 8, and >8 were excluded. Dynamic exclusion was enabled for 20 s and 30 s.

Garcia-Garcia T., Poncet S., Cuenot E., Douché T., Giai Gianetto Q., Peltier J., Courtin P., Chapot-Chartier M.P., Matondo M., Dupuy B., Candela T, & Martin-Verstraete I. (2021). Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile. mBio, 12(3), e00519-21.

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Mass Spectrometry Workflow for Proteomic Analysis

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An LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) was used for preliminary experiments. The ion transfer tube temperature was held at 300 °C. The S-Lens RF level was 50.00. The mass spectrometer was programmed in data-dependent mode. A top 20 method was used. Full MS scans were acquired with the Orbitrap mass analyzer over m/z 380–1,800 range with resolution of 70,000 (m/z 200) and the number of microscans set to 1. The target value was 1.00E+06, and maximum injection time was 250 ms. For MS/MS scans, the twenty most intense peaks with charge state ≥2 were sequentially isolated and further fragmented in the collision-induced dissociation (CID) mode following one full MS scan. The normalized collision energy was set at 30.
In a second experiment, a Q Exactive HF instrument (Thermo Scientific) was used with a 1-m long separation capillary. The mass spectrometer was programmed in data-dependent mode. A top 20 method was used. The S-lens RF level was set at 50, and heated capillary at 275 °C. Full scan resolution was set to 60,000 at m/z 200. Full scan target was 3.00E+06 with a maximum fill time of 30 ms. Mass range was set to 350–1,500. Target value for fragment scans was set at 1.00E+05, and intensity threshold was kept at 1.00E+05. Isolation width was set at 1.4 Th. A fixed first mass of 100 was used. Normalized collision energy was set at 28.

Zhang Z., Sun L., Zhu G., Cox O.F., Huber P, & Dovichi N.J. (2015). Nearly 1,000 protein identifications from 50 ng of Xenopus laevis zygote homogenate using on-line sample preparation on a strong cation exchange monolith-based microreactor coupled with capillary zone electrophoresis. Analytical chemistry, 88(1), 877-882.

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Comprehensive Proteomic Profiling of Human Cell Lines

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We used two types of human cell line data sets. The first type is global profiling datasets—11 human cell line datasets and HEK293 dataset. 11 human cell line data sets (PRIDE ID: PXD002395) was studied by Geiger and colleagues, acquired using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) coupled with high performance liquid chromatography (HPLC). The MS/MS scans obtained from 11 human cell lysates—A549, HEK293, GAMG, HeLa, HepG2, Jurkat, K562, MCF7, RKO, and U2OS cells, were composed of 136,309, 148,800, 152,777, 159,455, 149,974, 160,225, 167,429, 174,709, 164,317, 161,334, and 165,271 scans respectively. The second data set was high-throughput HEK293 data, composed of 1,121,149 scans generated by a Q-Exactive Orbitrap mass spectrometer [24 (link)] (PRIDE ID: PXD001468). The second type is the phosphorylation enrichment dataset. We used the human epithelial cervix carcinoma Hela cells (female), which was studied by Bekker-Jensen and colleagues [25 (link)]. The Hela phosphorylation data were analyzed on an EASY-nLC 1000 coupled to a Q-Exactive HF instrument (Thermo Fisher Scientific), coupled with a high pH reversed-phase HPLC fraction. The number of MS/MS spectra obtained from the Hela dataset was 362,356.

Li H., Na S., Hwang K.B, & Paek E. (2022). TIDD: tool-independent and data-dependent machine learning for peptide identification. BMC Bioinformatics, 23, 109.

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LC-MS/MS Analysis of Tryptic Peptides

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All LC-MS/MS analyses were executed on an Ultimate 3000 RSLCnano HPLC (Dionex, Idstein, Germany), which was coupled to a Q Exactive HF instrument (Thermo Fisher Scientific, Waltham, MA, USA). The measurements were carried out in random order. Of all samples, 300 ng of tryptic peptides was applied per measurement except for phosphoproteome analysis, where the entire eluted sample was used. Sample pre-concentration was achieved on a C18 trap column (Acclaim PepMap 100; 100 μm × 2 cm, 5 μm, 100 Å) in a period of 7 min at a flow rate of 30 μL/min using 0.1% TFA, and peptides were then transferred to a Nano Viper C18 analytical column (Acclaim PepMap RSLC; 75 μm × 50 cm, 2 μm, 100 Å). Separation of peptides was performed by applying a gradient from 5–40% solvent B over 120 min at 400 nL/min and 60 °C (solvent A: 0.1% formic acid (FA); solvent B: 0.1% FA, 84% ACN). For all samples, full-scan mass spectra were acquired in the Orbitrap analyzer in profile mode at a resolution of 60,000 at 400 m/z and within a mass range of 350–1400 m/z. Data-dependent mode at a resolution of 30,000 was utilized. The 10 most abundant ions per scan were selected for the MS/MS measurements and fragmented with higher-energy collision-induced dissociation (HCD; normalized collision energy (NCE) = 28).

Funke L., Bracht T., Oeck S., Schork K., Stepath M., Dreesmann S., Eisenacher M., Sitek B, & Schramm A. (2021). NTRK1/TrkA Signaling in Neuroblastoma Cells Induces Nuclear Reorganization and Intra-Nuclear Aggregation of Lamin A/C. Cancers, 13(21), 5293.

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Comprehensive Protein Identification Workflow

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Protein preparation, LC-MS/MS analysis, and a database search for the identification of proteins were essentially performed as previously described (62 (link)), except for the following changes. The LC gradient elution was as follows: 0 min at 4% eluent B, 5 min at 5% eluent B, 30 min at 8% eluent B, 60 min at 12% eluent B, 100 min at 20% eluent B, 120 min at 25% eluent B, 140 min at 35% eluent B, 150 min at 45% eluent B, 160 min at 60% eluent B, 170 to 175 min at 96% eluent B, and 175.1 to 200 min at 4% eluent B. Mass spectrometry analysis was performed on a QExactive HF instrument (Thermo Fisher Scientific) at a resolution of 120,000 FWHM for MS1 scans and 15,000 FWHM for MS2 scans. Tandem mass spectra were searched against the UniProt database (7 August 2018; https://www.uniprot.org/proteomes/UP000002530) of Neosartorya fumigata (Af293) and the human protein sequences of azurocidin, cathepsin G, and RBP7, using Proteome Discoverer (PD) software (version 2.2; Thermo Fisher Scientific) and the algorithms of Sequest HT (a version of PD software [version 2.2]) and MS Amanda (version 2.0) software. Modifications were defined as dynamic Met oxidation and protein N-terminal acetylation as well as static Cys carbamidomethylation.

Shopova I.A., Belyaev I., Dasari P., Jahreis S., Stroe M.C., Cseresnyés Z., Zimmermann A.K., Medyukhina A., Svensson C.M., Krüger T., Szeifert V., Nietzsche S., Conrad T., Blango M.G., Kniemeyer O., von Lilienfeld-Toal M., Zipfel P.F., Ligeti E., Figge M.T, & Brakhage A.A. (2020). Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus. mBio, 11(2), e00596-20.

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