Computational protocol: Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival‡

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Protocol publication

[…] Acquired MS spectra were processed with MaxQuant software package version (, ), integrated with the Andromeda search engine. Database search was performed against a target-decoy database of B. subtilis 168 obtained from Uniprot (taxonomy ID 1423), containing 4,195 B. subtilis protein entries and 248 commonly observed laboratory contaminant proteins. Endoprotease Lys-C was specified as the protease with a maximum missed cleavage of two. Three isotopic forms of lysine (light, medium, and heavy) were specified in the search space. Oxidation of methionines, N-terminal acetylation, and phosphorylation on serine, threonine, and tyrosine residues was specified as a variable modification. Initial maximum allowance for mass tolerance was set to six ppm for the survey scan and 0.5 Da for CID fragment ions. Carbamidomethylation on cysteines was set as a fixed modification. Re-quantify was enabled. A false discovery rate of 1% was applied at the peptide, protein and phosphorylated site level individually. Fragments with a minimum length of seven amino acids were used for SILAC peptide quantification. Phosphorylation events with a localization probability of ≥0.75 were considered localized on the respective S/T/Y residue. A posterior error probability (PEP) score filter of ≤0.001 was applied for the modified peptides. MS/MS spectra of phosphorylated peptides were manually validated for good b- and y- ion series coverage using MaxQuant Viewer (version Ratios from each SILAC experiment are relative to the Light labeled WT (control) strain. The SILAC ratios of the phosphorylation sites were further normalized to the respective protein ratios in order to eliminate a bias due to changing protein abundance. Phosphorylation sites were considered as differentially regulated based on “Significance B” (p = 0.05; ) calculated after normalization with the corresponding protein ratios. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Bacillus subtilis
Diseases Machado-Joseph Disease
Chemicals Adenosine Triphosphate, Amino Acids, Tyrosine