To identify phosphorylation sites of METTL3, METTL3 precipitated from 293T cells co-transfected with myc-METTL3 and B-Raf V600E was analyzed by Coomassie Blue staining. The protein band corresponding to METTL3 was excised and subjected to in-gel digestion with tryspin and chymotrypsin. Samples were analyzed by Ultimate Capillary LC system (Dionex) directly coupled to LTQ Orbitrap Mass Analyzer (Thermo Scientific) using the TopTenTM method. The data were searched on MASCOT (MassMatrix) against the human Swiss-Prot database. All the identified phospho-peptides were further confirmed by manually checking the results.
Ltq orbitrap mass analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LTQ Orbitrap Mass Analyzer is a high-resolution, high-accuracy mass spectrometer designed for analytical applications. It combines the sensitivity and speed of a linear ion trap with the high mass resolution and mass accuracy of an Orbitrap mass analyzer. The instrument is capable of providing precise and reliable mass measurements for a wide range of analytes.
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2 protocols using ltq orbitrap mass analyzer
1
Phosphorylation Site Identification of METTL3
2
High-Resolution Mass Spectrometry for Natural Product Characterization
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High-resolution mass spectrometry (MS) data were obtained using an LTQ Orbitrap mass analyzer coupled to an HPLC system (Photo diode array (PDA) detector, PDA Autosampler, and pump) (Thermo Fisher Scientific, Massachusetts, USA). The following conditions were used: capillary voltage, 45 V; capillary temperature, 260°C; auxiliary gas flow rate, 10−20 arbitrary units; sheath gas flow rate, 40−50 arbitrary units; spray voltage, 4.5 kV, and mass range 100−2000 emu (maximal resolution of 30,000). For LC-HRMS analysis, a SunFire C18 Analytical HPLC Column (5 μm, 4.6 × 150 mm (Waters™, Waters Corporation, Milford, Massachusetts, United States)) was used with a mobile phase of 0%–100% methanol (MeOH) over 30 min at a flow rate of 1 mL min−1. Data analysis was performed using Xcalibur 3.0 (Thermo Fisher Scientific, Massachusetts, USA) and dereplication was carried out using the Dictionary of Natural Products Database v.23.1.
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