Computational protocol: New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

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Protocol publication

[…] Coomassie-stained proteins were excised and subjected to tryptic in gel digestion [, ]. In brief, proteins were reduced with 20 mM DTT, slices washed with 100 mM ammonium bicarbonate, and proteins alkylated with 40 mM iodoacetamide. The slices were washed again and dehydrated with acetonitrile. Gel pieces were dried in a vacuum concentrator and incubated with 400 ng sequencing grade trypsin at 37°C overnight. The peptide extract was dried in a vacuum concentrator and stored at -20°C. Dried peptides were dissolved in 8 μl 0.1% formic acid (solvent A). 2 μl were injected onto a C18 trap column (20 mm length, 100 μm inner diameter). Bound peptides were eluted onto a C18 analytical column (200 mm length, 75 μm inner diameter, both columns from NanoSeparations). Peptides were separated during a linear gradient from 1% to 45% solvent B (80% acetonitrile, 0.1% formic acid) within 18 min at a flow rate of 450 nl/min. The nano-HPLC was coupled online to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptide ions between 330 and 2000 m/z were scanned in the Orbitrap detector with a resolution of 30,000 (maximum fill time 400 ms, AGC target 106). The eight most intense precursor ions (threshold intensity 5000) were subjected to collision induced dissociation with multiple stage activation (MSA, normalized collision energy 35, wide band activation) and fragment spectra recorded in the linear ion trap. After acquisition of eight MSA-spectra, the same eight precursor ions were subjected to higher energy collisional dissociation (HCD, normalized collision energy 42) and fragment spectra recorded in the Orbitrap detector. Fragmented peptide ions were excluded from repeat analysis for 20 s. Raw data processing and analysis of database searches were performed with Proteome Discoverer software 1.3 (Thermo Fisher Scientific). Peptide identification was done with an in house Mascot server version 2.3 (Matrix Science Ltd) and the Sequest search node in Proteome Discoverer. MS2 data were searched against mouse sequences from SwissProt (release 2012_07). Precursor ion m/z tolerance was 8 ppm, fragment ion tolerance 0.6 Da (CID) or 0.02Da (HCD). b- and y-ion series were included. Semitryptic peptides with up to two missed cleavages were searched. The following dynamic modifications were tested: phosphorylation of serine, threonine, and tyrosine, oxidation of methionine, propionamide or carbamidomethyl on cysteine, and acetylation of the protein N-terminus. Mascot and Sequest results from searches against SwissProt were sent to the percolator algorithm version [] 1.17 as implemented in Proteome Discoverer. The PhosphoRS2.0 node was used for scoring of the phosphosite assignment []. Spectra with low scoring identifications and phospho-localizations were inspected manually. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server, Comet, ptmRS
Application MS-based untargeted proteomics
Organisms Mus musculus
Chemicals Calcium, Kainic Acid