Computational protocol: Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC‐A

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Protocol publication

[…] Microsomes from WT and Tric‐a KO mouse skeletal muscle were treated with PKA as described previously (Carter et al. ). Microsomal proteins were separated on a 6% SDS‐PAGE and either stained with Coomassie Brilliant Blue for visualization or transferred to a nitrocellulose membrane and probed with RyR1 antibody as described above. The corresponding bands containing RyR1 were cut from the Coomassie Brilliant Blue stained gel and subjected to in‐gel tryptic digestion for MS analysis as described previously (Shevchenko et al. ). The peptides generated were then separated by nanoflow reversed‐phase liquid chromatography coupled to Q Exactive Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were loaded on a C18 PepMap100 pre‐column (inner diameter 300 μm × 5 mm, 3 μm C18 beads; Thermo Fisher Scientific) and separated on a 50 cm reversed‐phase C18 column (inner diameter 75 μm, 2 μm C18 beads). Separation was conducted with a linear gradient of 7–30% of B for 30 min at a flow rate of 200 nl min−1 (A: 0.1% formic acid, B: 0.1% formic acid in acetonitrile). All data were acquired in a data‐dependent mode, automatically switching from MS to collision‐induced dissociation MS/MS on the top 10 most abundant ions with a precursor scan range of 350–1650 m/z. MS spectra were acquired at a resolution of 70 000 and MS/MS scans at 17 000. Dynamic exclusion was enabled with an exclusion duration of 40 s. The raw data files generated were processed using MaxQuant, version, integrated with the Andromeda search engine as described previously (Cox & Mann ; Cox et al. ). The MS/MS spectra were searched against the mouse proteome (UniProt 2013/04/03), precursor mass tolerance was set to 20 ppm with variable modifications defined as phosphorylation (S, T and Y). Enzyme specificity was set to trypsin with a maximum of two missed cleavages. Protein and peptide spectral matches false discovery rate was set at 0.01 and a minimum score of 40 and localization probability of > 0.7 for phosphopeptides. Match between runs was applied. The ratio of phosphorylated (S2844) to unphosphorylated KISQTAQTYDPR peptide intensities was calculated for three biological replicates of WT and Tric‐a KO under control and PKA treatment. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Heart Diseases
Chemicals Adenine, Adenosine Triphosphate, Caffeine, Ryanodine