To evaluate TRIB2 binding in vitro, afatinib was incubated for 15 min with purified 6His-TRIB2 at a 1:10 molar ratio, then denatured with 0.05% (w/v) RapiGest SF (Waters Corporation), and digested with chymotrypsin [1:20 protease/protein (w/w) ratio] for 16 hours at 25°C. RapiGest hydrolysis was induced by the addition of triflouroacetic acid (TFA) to 1% (v/v), incubated at 37°C for 1 hour. Insoluble product was removed by centrifugation (13,000g, 20 min). Reversed-phase high-performance liquid chromatography separation was performed using an UltiMate 3000 nano system (Dionex) coupled in-line with a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Digested peptides (500 fmol) were loaded onto the trapping column (PepMap100 C18, 300 μm × 5 mm), using partial loop injection, for 7 min at a flow rate of 9 μl/min with 2% (v/v) MeCN and 0.1% (v/v) TFA and then resolved on an analytical column (Easy-Spray C18, 75 μm × 500 mm, 2-μm bead diameter column) using a gradient of 96.2% A [0.1% (v/v) formic acid (FA)]/3.8% B [80% (v/v) MeCN, 0.1% (v/v) FA] to 50% B over 35 min at a flow rate of 300 nl/min. MS1 spectra were acquired over m/z 400 to 1500 in the orbitrap (60 K resolution at m/z 200). Data-dependent MS2 analysis was performed using a top-speed approach (cycle time of 3 s), using higher-energy collisional dissociation and electron-transfer and higher-energy collision dissociation for fragmentation, with product ions being detected in the ion trap (rapid mode). Data were processed using Thermo Proteome Discoverer (v. 1.4), and spectra were searched in MASCOT against the Escherichia coli International Protein Index database with the added sequence of full-length 6His-TRIB2 (1 to 343). Parameters were set as follows: MS1 tolerance of 10 ppm, MS/MS mass tolerance of 0.6 Da, with oxidation of methionine and afatinib binding at cysteine as variable modifications. MS2 spectra were interrogated manually.

To evaluate the interaction between afatinib and intact TRIB2 protein, TRIB2 was incubated with afatinib as above and then desalted using a C4 desalting trap (Waters MassPREP Micro desalting column, 2.1 mm × 5 mm, 20 μm particle size, 1000 Å pore size). TRIB2 was eluted with 50% (v/v) MeCN and 0.1% (v/v) FA. Intact TRIB2 mass analysis was performed using a Waters nanoACQUITY UPLC (ultra performance liquid chromatography) system coupled to a Waters SYNAPT G2. Samples were eluted from a C4 trap column at a flow rate of 10 μl/min using three repeated 0 to 100% acetonitrile gradients. Data were collected between m/z 400 and 3500 and processed using MaxEnt1 (maximum entropy software, Waters Corporation).

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