In vitro kinase assays were performed by incubating 75 ng CDK1/Cyclin B recombinant human protein (Invitrogen, Waltham, MA) and 100 μg TFCP2L1 wild‐type (WT; IQVHCISTEFTPRKHGGEK) or T177A variant (IQVHCISTEFAPRK HGGEK) peptides (Peptron, Daejeon, Korea) in kinase buffer [60 mM HEPES‐NaOH pH 7.5, 3 mM MgCl2, 3 mM Na‐orthovanadate, 1.2 mM DTT, and 0.5 mM ATP] (Cell Signaling Technology) at 30°C for 15 min. The peptide mixture from the in vitro kinase assay was desalted using C18 reverse phase chromatography using ZipTip (Merck, Germany) and dried. The reconstituted peptide mixture containing 0.1% formic acid was introduced into the Ultimate 3000 RSLCnano system (Thermo Fisher Scientific), and the mass to charge ratio of the ionized peptide was measured using Q Exactive Plus Orbitrap mass spectrometry (Thermo Fisher Scientific). LC, mass spectrometry, and database searches were performed as described previously.
Q exactive plus orbitrap mass spectrometry
Manufactured by Thermo Fisher Scientific
The Q Exactive Plus Orbitrap mass spectrometry is a high-resolution, high-mass-accuracy mass spectrometer. It combines a quadrupole mass filter with an Orbitrap mass analyzer to provide accurate mass measurements and high-resolution data acquisition.
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2 protocols using q exactive plus orbitrap mass spectrometry
1
In vitro Phosphorylation of TFCP2L1
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In vitro kinase assays were performed by incubating 75 ng CDK1/Cyclin B recombinant human protein (Invitrogen, Waltham, MA) and 100 μg TFCP2L1 wild‐type (WT; IQVHCISTEFTPRKHGGEK) or T177A variant (IQVHCISTEFAPRK HGGEK) peptides (Peptron, Daejeon, Korea) in kinase buffer [60 mM HEPES‐NaOH pH 7.5, 3 mM MgCl2, 3 mM Na‐orthovanadate, 1.2 mM DTT, and 0.5 mM ATP] (Cell Signaling Technology) at 30°C for 15 min. The peptide mixture from the in vitro kinase assay was desalted using C18 reverse phase chromatography using ZipTip (Merck, Germany) and dried. The reconstituted peptide mixture containing 0.1% formic acid was introduced into the Ultimate 3000 RSLCnano system (Thermo Fisher Scientific), and the mass to charge ratio of the ionized peptide was measured using Q Exactive Plus Orbitrap mass spectrometry (Thermo Fisher Scientific). LC, mass spectrometry, and database searches were performed as described previously.
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In vitro kinase assays were performed by incubating 75 ng CDK1/Cyclin B recombinant human protein (Invitrogen, Waltham, MA) and 100 μg TFCP2L1 wild‐type (WT; IQVHCISTEFTPRKHGGEK) or T177A variant (IQVHCISTEFAPRK HGGEK) peptides (Peptron, Daejeon, Korea) in kinase buffer [60 mM HEPES‐NaOH pH 7.5, 3 mM MgCl2, 3 mM Na‐orthovanadate, 1.2 mM DTT, and 0.5 mM ATP] (Cell Signaling Technology) at 30°C for 15 min. The peptide mixture from the in vitro kinase assay was desalted using C18 reverse phase chromatography using ZipTip (Merck, Germany) and dried. The reconstituted peptide mixture containing 0.1% formic acid was introduced into the Ultimate 3000 RSLCnano system (Thermo Fisher Scientific), and the mass to charge ratio of the ionized peptide was measured using Q Exactive Plus Orbitrap mass spectrometry (Thermo Fisher Scientific). LC, mass spectrometry, and database searches were performed as described previously.
2
High-Resolution Peptide Separation and Analysis
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The Easy nLC 1000 system (Thermo Scientific, Rockford, IL) was used to separate the peptides. Dried peptides were resuspended in mobile phase A (0.1% formic acid in water). Then, 4 μL of the sample was injected and separated on a C18 analytical column (75 μm × 100 nm, 3 μm) with a linear gradient elution of mobile phase B. The mobile phase B was 0.1% formic acid in 80% acetonitrile. The flow rate was set at 300 nL/min. Q-Exactive-Plus Orbitrap Mass Spectrometry (Thermo Scientific) was used to analyze the separated peptides under the following conditions: the polarity was positive and default charge state was 2. Full MS was scanned from 350 to 2,000 mass/charge (m/z) at a resolution of 70,000, and the maximum ion injection time was 50 ms. The resolution for data-dependent MS/MS spectra was set at 17,500 with a fixed first mass of 110 m/z. The normalized collision energy was 32 eV and the dynamic exclusion was 60 s.
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