PRM analysis was performed using QExactive HF mass spectrometer equipped with EASY-nano LC 1000 (Thermo Scientific). Enriched immunopeptide samples spiked with stable isotope labeled peptides were loaded onto a trap column (Acclaim PepMap 100, 2 cm × 75 μm, Thermo fisher) and PepMap RSLC C18 50 cm × 75 μm analytical column (Thermo Fisher). Peptides were separated using the low pH mobile phases (A: 0.1% FA in water; B: 0.1% FA in ACN) with a flow rate of 300 nL per min and a linear gradient of 3–7% buffer B in 0–3 min, 30% in 103 min, 40% in 106 min, and an increase to 75% in 111 min with a hold for 6 min before returning to the initial condition of 3% buffer B. Peptide samples were analyzed using the PRM method based on scheduled inclusion lists of HLA-I and HLA-II peptides. The PRM scan events collected using an Orbitrap mass resolution of 30,000, an AGC value of 1 × 106, and maximum injection time of 250 ms with an isolation width of 1 m/z. Fragmentation was performed with a normalized collision energy of 28.
Q exactive hf mass
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Q Exactive HF mass spectrometer is a high-resolution, high-mass-accuracy mass spectrometer designed for advanced proteomics and metabolomics applications. It features a quadrupole-Orbitrap hybrid mass analyzer that provides high-quality data for both targeted and untargeted analysis.
Automatically generated - may contain errors
Lab products found in correlation
Easy nanolc 1000, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (1 mentions)
Sep pak c18, by Waters Corporation (1 mentions)
Easy nlc, by Thermo Fisher Scientific (1 mentions)
C18 beads, by Dr. Maisch (1 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (1 mentions)
5 protocols using q exactive hf mass
1
PRM Analysis of HLA-I and HLA-II Peptides
2
LC/MS Analysis of Biological Samples
3
Proteomic Analysis of Protein Samples
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The stained protein lane of the gel was excised in its entirety, reduced with TCEP, alkylated with iodoacetamide, and digested with trypsin. Resulting digests were analyzed using a 90-min LC gradient on the Thermo Q Exactive HF mass spectrometer. Protein quantification was performed using shared (Razor)+unique peptides. Razor peptides were assigned to the protein group with the most other peptides. Proteins identified by a single razor+unique peptide were considered low confident identifications and removed from the dataset.
4
Polysome Fraction Proteomic Analysis
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For mass spectrometry sample preparation, the polysome containing fractions were diluted 2× in 6 M guanidinium chloride, 75 mM NaCl, 100 mM Tris–HCl, pH 8.5, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 10 mM chloroacetamide (CAA) and sequentially digested with Lys-C and trypsin. The resulting peptides were desalted on C18 Sep-Pak (Waters) columns. The eluates from Sep-Pak columns were reconstituted in 0.1% trifluoroacetic acid and 5% acetonitrile. All samples were analyzed using a Q-Exactive HF mass spectrometer connected to an EASY-nLC (Thermo Fisher Scientific). Peptides were chromatographically separated on a 15 cm long column, 75-μm inner diameter, packed in-house with 1.9 μm C18 beads (Reprosil-Pur AQ, Dr Maisch) using a 60 min gradient. The mass spectrometer was operated in a data-dependent acquisition mode using a top-6 method. The resolution for full scans was set to 60 000 and peptides dissociated using HCD were analyzed at a 30,000 resolution. All raw mass spectrometry output files were analyzed using MaxQuant (version 1.6.6.0) and subsequent statistical analysis was carried out using Perseus (version 1.6.5.0).
5
Targeted Quantification of Proteins
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Protein extraction and tryptic digestion were conducted as previously described for the SWATH.[50 ] Forty target proteins for PRM validation were analyzed on an Easy‐nLC 1200 system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A scheduled PRM method consisting of full MS1 scan and targeted MS2 scans was developed. The full MS1 scan was collected from 350 to 1650 m/z at a resolution of 120 000 (AGC target: 3e6, maximum injection time: 100 ms). Targeted MS2 scans were collected from 200 to 2000 m/z at a resolution of 30 000 (AGC target: 1e5, maximum injection time: 80 ms). Precursors were isolated within a 1.2 m/z window and fragmented with a normalized collision energy of 27. The peptides monitored for each protein were imported to the Skyline software, and the peptides for protein quantification were selected according to the ion signals in spectra library. Data processing was performed in Skyline, and the quantification results were manually inspected for each peptide of the targeted proteins.
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