Computational protocol: Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA

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Protocol publication

[…] Following SDS-PAGE analysis of Dvl3 immunocomplexes, the gel band corresponding to the molecular range of 50–80 kDa was excised, destained, reduced, aklyated and digested with either trypsin or chymotrypsin (Promega Gold, Mass Spectrometry Grade) essentially as described previously () with minor modifications. The resulting concentrated peptide extract was diluted into a solution of 2% acetonitrile (ACN), 0.1% formic acid (FA) (Buffer A) for analysis. 10 μl of the peptide mixture was analyzed by automated microcapillary liquid chromatography tandem mass spectrometry. Fused-silica capillaries (100 μm i.d.) were pulled using a P-2000 CO2 laser puller (Sutter Instruments, Novato, CA) to a 5 μm i.d. tip and packed with 10 cm of 5 μm Magic C18 material (Agilent, Santa Clara, CA) using a pressure bomb. This column was then placed in-line with a Dionex 3000 HPLC equipped with an autosampler. The column was equilibrated in buffer A, and the peptide mixture was loaded onto the column using the autosampler. The HPLC separation at a flow rate of 300 nl/minute was provided by a gradient between Buffer A and Buffer B (98% acetonitrile, 0.1% formic acid). The HPLC gradient was held constant at 100% buffer A for 5 minutes after peptide loading followed by a 30 minute gradient from 5% buffer B to 40% buffer B. Then, the gradient was switched from 40% to 80% buffer B over 5 minutes and held constant for 3 minutes. Finally, the gradient was changed from 80% buffer B to 100% buffer A over 1 minute, and then held constant at 100% buffer A for 15 more minutes. The application of a 1.8 kV distal voltage electrosprayed the eluted peptides directly into a Thermo LTQ ion trap mass spectrometer equipped with a custom nanoLC electrospray ionization source. Full mass (MS) spectra were recorded on the peptides over a 400–2000 m/z range, followed by five tandem mass (MS/MS) events sequentially generated in a data-dependent manner on the first, second, third, fourth and fifth most intense ions selected from the full MS spectrum (at 35% collision energy). Mass spectrometer scan functions and HPLC solvent gradients were controlled by the Xcalibur data system (ThermoFinnigan, San Jose, CA). MS/MS spectra were extracted from the RAW file with ReAdW.exe (Sourceforge). The resulting mz XML file contains all the data for all MS/MS spectra and can be read by the subsequent analysis software. The MS/MS data was searched with Inspect () against a mouse database (IPI v.3.43) plus common contaminants, with modifications: +16 on Met, +57 on Cys, +14 on Arg and Lys. Only peptides with a P value of at least 0.02 were analyzed further. Peptides with possible methylated arginines and lysines were manually verified. […]

Pipeline specifications

Software tools ReAdW, Inspect
Application MS-based untargeted proteomics
Organisms Mus musculus, Homo sapiens